Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.
SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.
SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.SEAP Activity Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 ul of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. After 6-8 hours of stimulation with test compounds and PGE2, 10 ul of culture media from each well was transferred to a corresponding well of a 96-well solid black plate. Cover the plate with the lid.
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assayAgonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assay
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assayAgonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assay
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assayAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in GalphaS-mediated CREB activation measured after 6 to 24 hrs by SEAP reporter gene-based chemiluminescence assay
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
Agonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF methodAgonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF method
Agonist activity at rat EP4 receptor expressed in HEK293 cells assessed as cAMP activationAgonist activity at rat EP4 receptor expressed in HEK293 cells assessed as cAMP activation
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity against rat EP4 receptor expressed in HEK293 cells assessed as stimulation of cAMP releaseAgonist activity against rat EP4 receptor expressed in HEK293 cells assessed as stimulation of cAMP release
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assayAgonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as potentiation of PGE2-induced cAMP accumulation by scintillation proximity assay
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: EP4 receptors couple to Gs and mediate elevations in cAMP concentration, although they do participate in other pathways as well. There are some redundancies in function between EP2 and EP4 receptors. For example, both receptors induce PGE2-mediated RANKL through cAMP.cAMP Assay: EP4 receptors couple to Gs and mediate elevations in cAMP concentration, although they do participate in other pathways as well. There are some redundancies in function between EP2 and EP4 receptors. For example, both receptors induce PGE2-mediated RANKL through cAMP.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at mouse EP4 receptor expressed in CHO cells assessed as cAMP productionAgonist activity at mouse EP4 receptor expressed in CHO cells assessed as cAMP production
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Agonist activity at human recombinant EP4 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP4 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Agonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF methodAgonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF method
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Agonist activity at rabbit EP4 receptor assessed as relaxation of Kcl-induced tissue contraction by isometric transducer methodAgonist activity at rabbit EP4 receptor assessed as relaxation of Kcl-induced tissue contraction by isometric transducer method
Agonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISAAgonist activity at human EP4 receptor expressed in HEK293T/17 cells assessed as increase in intracellular cAMP level incubated for 30 mins by ELISA
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Agonist activity at human recombinant EP4 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assayAgonist activity at human recombinant EP4 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Agonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF methodAgonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF method
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Agonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulationAgonist activity at EP4 receptor expressed in HEK293 cells assessed as cAMP accumulation
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptorEffective concentration for increased intracellular c-AMP production by mouse Prostanoid EP4 receptor
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF methodAgonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF method
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at rabbit EP4 receptor assessed as relaxation of Kcl-induced tissue contraction by isometric transducer methodAgonist activity at rabbit EP4 receptor assessed as relaxation of Kcl-induced tissue contraction by isometric transducer method
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10N HCl is added to achieve a pH of 7.4). The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 °C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2a (5 nM) were performed in a 100 μl volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell harvester. The filters were washed 3 times with ice-cold buffer and oven dried for one hour. [3H−] PGE2 (specific activity 180 Ci mmol) was used as the radioligand for EP receptors. [3H] 17-phenyl PGF2a was employed for FP receptor binding studies. Binding studies employing EP1, EP2, EP4 and FP receptors were performed in duplicate in at least three separate experiments. A 200 μl assay volume was used. Incubations were for 60 min at 25 °C. and were terminated by the addition of 4 ml of ice-cold 50 mM TRIS-HCl, followed by rapid filtration through Whatman GF/B filters and three additional 4 ml washes in a cell harvester (Brandel). Competition studies were performed using a final concentration of 5 nM [3H]-PGE2, or 5 nM [3H] 17-phenyl PGF2a and non-specific binding determined with 10^−5M of unlabeled PGE2, or 17-phenyl PGF2a, according to receptor subtype studied.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10N HCl is added to achieve a pH of 7.4). The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 °C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2a (5 nM) were performed in a 100 μl volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell harvester. The filters were washed 3 times with ice-cold buffer and oven dried for one hour. [3H−] PGE2 (specific activity 180 Ci mmol) was used as the radioligand for EP receptors. [3H] 17-phenyl PGF2a was employed for FP receptor binding studies. Binding studies employing EP1, EP2, EP4 and FP receptors were performed in duplicate in at least three separate experiments. A 200 μl assay volume was used. Incubations were for 60 min at 25 °C. and were terminated by the addition of 4 ml of ice-cold 50 mM TRIS-HCl, followed by rapid filtration through Whatman GF/B filters and three additional 4 ml washes in a cell harvester (Brandel). Competition studies were performed using a final concentration of 5 nM [3H]-PGE2, or 5 nM [3H] 17-phenyl PGF2a and non-specific binding determined with 10^−5M of unlabeled PGE2, or 17-phenyl PGF2a, according to receptor subtype studied.
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Partial agonist activity at EP4 receptor in human whole blood assessed as inhibition of LPS-induced TNFalpha productionPartial agonist activity at EP4 receptor in human whole blood assessed as inhibition of LPS-induced TNFalpha production
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Effective concentration required for prostanoid EP4 receptor activity was determinedEffective concentration required for prostanoid EP4 receptor activity was determined
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Agonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assayAgonist activity at human EP4 receptor expressed in HEK293 cells by calcium-5 dye based FLIPR assay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF methodAgonist activity at human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level after 30 mins by HTRF method
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
Effective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptorEffective concentration which increases intracellular c-AMP production in mouse Prostanoid EP4 receptor
Partial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assayPartial agonist activity at human EP4 receptor expressed in HEK293-AEQ17 cells assessed as cAMP accumulation by aequorin luminescence assay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.cAMP Assay: A 384-well drug plate was prepared to contain test compounds, PGE2 and cAMP in 16 serial dilutions in triplicate, using a Biomek station. HEK-EBNA cells expressing a target PG receptor subtype (EP2 or EP4) were suspended in a stimulation buffer (HBSS, 0.1% BSA, 0.5 mM IBMX and 5 mM HEPES, pH 7.4) in a density of 104 cells/5 μl. The reaction was initiated by mixing 5 μL drug dilutions with 5 μl of HEK-EBNA cells in a well, carried out for 30 min at room temperature, and followed by the addition of 5 μl anti-cAMP acceptor beads in the control buffer with Tween-20 (25 mM NaCl, 0.03% Tween-20, 5 mM HEPES, pH7.4). After 30 min in the dark at room temperature, the mixtures were incubated with 15 μl biotinylated-cAMP/strpavidin donor beads in Lysis/Detection buffer (0.1% BSA, 0.3% Tween-20 and 5 mM HEPES, pH7.4) for 45 min at the room temperature. Fluorescence changes were read using a Fusion-alpha HT microplate reader.
cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.cAMP Assay: 1. Seed cells on an EP2 or EP4 STEP plate at a density of 40,000-80,000 cells/well in 200 uL of reduced serum medium containing 0.5% FBS. Place the plate in a 37° C. incubator with 5% CO2 and incubate overnight. 2. After 16-18 hours of incubation, aspirate the culture media from each well. 3. Add 200 ul of culture medium containing 500 uM IBMX (an inhibitor of cAMP phosphodiesterase) and different concentration of test compounds to the assigned wells. For each test compound, at least 8 concentrations starting at highest 10 M and lowest 0.01 pM were tested. In addition each concentration had triplicates. A PGE2 curve (concentrations from lowest to highest, 0 pM, 0.384 pM, 1.92 pM, 9.6 pM, 48 pM, 240 pM, 1200 pM, and 6000 pM) was always run in parallel with test compounds. 4. Incubate the cells in a cell culture incubator for 30 minutes. 5. Centrifuge the plate at 1,000x rpm for 10 minutes. 6. Aspirate the supernatant.
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cellsAgonist activity against recombinant prostanoid EP4 receptor stably transfected in CHO cells
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIAAgonist activity at recombinant human EP4 receptor expressed in CHO cells assessed as increase in intracellular cAMP level by EIA
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Agonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassayAgonist activity at rat EP4 receptor expressed in CHO cells assessed as cAMP production after 10 mins by radioimmunoassay
Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assay
Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).
Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).
Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).
Inhibition of human EP4 transfected in human HEK293 cells co transfected with SmBit-beta-arrestin. assessed as reduction in PGE2 induced-beta-arrestin recruitment by NanoBiT beta-arrestin recruitment assayInhibition of human EP4 transfected in human HEK293 cells co transfected with SmBit-beta-arrestin. assessed as reduction in PGE2 induced-beta-arrestin recruitment by NanoBiT beta-arrestin recruitment assay
Binding affinity to human prostanoid EP4 receptor by radioligand displacement assayBinding affinity to human prostanoid EP4 receptor by radioligand displacement assay
Displacement of [3H]PGE2 from human recombinant EP4 receptor expressed in HEK293 cells measured after 120 mins by scintillation counting methodDisplacement of [3H]PGE2 from human recombinant EP4 receptor expressed in HEK293 cells measured after 120 mins by scintillation counting method
Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assay
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assay
Inhibitory activity against human EP4 receptor expressed in HEK293 ebna cellsInhibitory activity against human EP4 receptor expressed in HEK293 ebna cells
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assay
Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Displacement of [3H]PGE2 from human recombinant prostanoid EP4 receptor in CHEM1 cells after 2 hrsDisplacement of [3H]PGE2 from human recombinant prostanoid EP4 receptor in CHEM1 cells after 2 hrs
Antagonist activity at human EP4 receptor in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulationAntagonist activity at human EP4 receptor in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Antagonist activity at human EP4 receptor in HEK293 cells by cell-based functional assayAntagonist activity at human EP4 receptor in HEK293 cells by cell-based functional assay
Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Inhibitory activity against human EP4 receptor expressed in HEK293 ebna cellsInhibitory activity against human EP4 receptor expressed in HEK293 ebna cells
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation countingDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assay
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Antagonist activity against human EP4 expressed in HEK293 cells assessed as inhibition of PGE2-stimulated production of cAMP incubated for 20 mins by HTRF assayAntagonist activity against human EP4 expressed in HEK293 cells assessed as inhibition of PGE2-stimulated production of cAMP incubated for 20 mins by HTRF assay
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay in presence of 10% human serumAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay in presence of 10% human serum
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assay
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serumDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serum
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by bead-based proximity assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by bead-based proximity assay
Displacement of [3H]PGE2 from human prostanoid EP4 receptor expressed in cell membranes by scintillation countingDisplacement of [3H]PGE2 from human prostanoid EP4 receptor expressed in cell membranes by scintillation counting
Displacement of [3H]PGE2 from human prostanoid EP4 receptor expressed in cell membranes by scintillation countingDisplacement of [3H]PGE2 from human prostanoid EP4 receptor expressed in cell membranes by scintillation counting
Displacement of [3H]PGE2 from human prostanoid EP4 receptor expressed in cell membranes by scintillation countingDisplacement of [3H]PGE2 from human prostanoid EP4 receptor expressed in cell membranes by scintillation counting
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assay
Displacement of [3H]PGE2 from human EP4 receptor after 1 hr by liquid scintillation countingDisplacement of [3H]PGE2 from human EP4 receptor after 1 hr by liquid scintillation counting
Antagonist activity at EP4 receptor in human whole blood assessed as blockade of inhibition of TNF-alpha-induced IP10 releaseAntagonist activity at EP4 receptor in human whole blood assessed as blockade of inhibition of TNF-alpha-induced IP10 release
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serumDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serum
Displacement of [3H]PGE from human EP4 receptor expressed in HEK293 cell membranesDisplacement of [3H]PGE from human EP4 receptor expressed in HEK293 cell membranes
Activity at EP4 receptor in human whole blood assessed as blockade of inhibition of TNF-alpha-induced IP10 releaseActivity at EP4 receptor in human whole blood assessed as blockade of inhibition of TNF-alpha-induced IP10 release
cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.
Antagonist activity at human EP4 receptor in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulationAntagonist activity at human EP4 receptor in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation
cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.
Antagonist activity against human EP4 expressed in HEK293 cells assessed as inhibition of PGE2-stimulated production of cAMP incubated for 20 mins by HTRF assayAntagonist activity against human EP4 expressed in HEK293 cells assessed as inhibition of PGE2-stimulated production of cAMP incubated for 20 mins by HTRF assay
Antagonist activity at human EP4 receptor in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulationAntagonist activity at human EP4 receptor in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation
Antagonist activity at recombinant human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP accumulation by scintillation proximity assay in presence of [125I]-cAMPAntagonist activity at recombinant human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP accumulation by scintillation proximity assay in presence of [125I]-cAMP
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Antagonist activity at mouse EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at mouse EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Antagonist activity against EP4 in human whole blood assessed as reversal of PGE2-mediated suppression of LPS-induced TNF-alpha production preincubated for 30 mins followed by LPS stimulation measured after 20 to 24 hrs by enzyme immunoassayAntagonist activity against EP4 in human whole blood assessed as reversal of PGE2-mediated suppression of LPS-induced TNF-alpha production preincubated for 30 mins followed by LPS stimulation measured after 20 to 24 hrs by enzyme immunoassay
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Antagonist activity against rat EP4 assessed as inhibition of PGE2-stimulated production of cAMPAntagonist activity against rat EP4 assessed as inhibition of PGE2-stimulated production of cAMP
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Antagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Displacement of [3H]PGE2 from human prostanoid EP4 receptor expressed in cell membranes by scintillation countingDisplacement of [3H]PGE2 from human prostanoid EP4 receptor expressed in cell membranes by scintillation counting
Antagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISAAntagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISA
Antagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISAAntagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISA
Antagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISAAntagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISA
Antagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISAAntagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISA
Antagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha release pretreated for 30 mins followed by addition of PGE2 measured after 20 to 24 hrs by immunoassayAntagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha release pretreated for 30 mins followed by addition of PGE2 measured after 20 to 24 hrs by immunoassay
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assay
Antagonist activity against EP4 in human whole blood assessed as reversal of PGE2-mediated suppression of LPS-induced TNF-alpha production preincubated for 30 mins followed by LPS stimulation measured after 20 to 24 hrs by enzyme immunoassayAntagonist activity against EP4 in human whole blood assessed as reversal of PGE2-mediated suppression of LPS-induced TNF-alpha production preincubated for 30 mins followed by LPS stimulation measured after 20 to 24 hrs by enzyme immunoassay
Antagonist activity at EP4 receptor in human whole blood assessed as reversal of PGE2 inhibitory effect on LPS-induced TNF-alpha production preincubated for 30 mins followed by LPS/PGE2 addition measured after 20 to 24 hrs by enzyme immunoassayAntagonist activity at EP4 receptor in human whole blood assessed as reversal of PGE2 inhibitory effect on LPS-induced TNF-alpha production preincubated for 30 mins followed by LPS/PGE2 addition measured after 20 to 24 hrs by enzyme immunoassay
Antagonist activity at EP4 receptor in human whole blood assessed as reversal of PGE2 inhibitory effect on LPS-induced TNF-alpha production preincubated for 30 mins followed by LPS/PGE2 addition measured after 20 to 24 hrs by enzyme immunoassayAntagonist activity at EP4 receptor in human whole blood assessed as reversal of PGE2 inhibitory effect on LPS-induced TNF-alpha production preincubated for 30 mins followed by LPS/PGE2 addition measured after 20 to 24 hrs by enzyme immunoassay
Displacement of [3H]PGE from human EP4 receptor expressed in HEK293 cell membranesDisplacement of [3H]PGE from human EP4 receptor expressed in HEK293 cell membranes
Antagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISAAntagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISA
Antagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISAAntagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISA
Antagonist activity at human EP4 receptor in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulationAntagonist activity at human EP4 receptor in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by bead-based proximity assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by bead-based proximity assay
Displacement of [3H]PGE from human EP4 receptor expressed in HEK293 cell membranesDisplacement of [3H]PGE from human EP4 receptor expressed in HEK293 cell membranes
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Antagonist activity at mouse EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at mouse EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Displacement of [3H]PGE2 from from human EP4 receptor expressed in HEK293 cells membranes incubated for 60 minsDisplacement of [3H]PGE2 from from human EP4 receptor expressed in HEK293 cells membranes incubated for 60 mins
Antagonist activity at mouse EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at mouse EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Antagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Antagonist activity at mouse EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at mouse EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Antagonist activity at EP4 receptor in human whole blood assessed as reversal of inhibition of PGE2 mediated LPS-induced TNF alpha production pretreated for 30 mins using 3,3',5,5' tetramethylbiphenyl-4,4'-diamine substrate measured after 20 to 24 hrs by immunoassayAntagonist activity at EP4 receptor in human whole blood assessed as reversal of inhibition of PGE2 mediated LPS-induced TNF alpha production pretreated for 30 mins using 3,3',5,5' tetramethylbiphenyl-4,4'-diamine substrate measured after 20 to 24 hrs by immunoassay
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assay
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serumDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serum
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serumDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serum
Antagonist activity against human EP4 expressed in HEK293 cells assessed as inhibition of PGE2-stimulated production of cAMP incubated for 20 mins by HTRF assayAntagonist activity against human EP4 expressed in HEK293 cells assessed as inhibition of PGE2-stimulated production of cAMP incubated for 20 mins by HTRF assay
Antagonist activity at recombinant human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP accumulation by scintillation proximity assay in presence of [125I]-cAMPAntagonist activity at recombinant human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP accumulation by scintillation proximity assay in presence of [125I]-cAMP
Antagonist activity at recombinant human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP accumulation by scintillation proximity assay in presence of [125I]-cAMPAntagonist activity at recombinant human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP accumulation by scintillation proximity assay in presence of [125I]-cAMP
Antagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Antagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assay
Antagonist activity against EP4 in human whole blood assessed as reversal of PGE2-mediated suppression of LPS-induced TNF-alpha production preincubated for 30 mins followed by LPS stimulation measured after 20 to 24 hrs by enzyme immunoassayAntagonist activity against EP4 in human whole blood assessed as reversal of PGE2-mediated suppression of LPS-induced TNF-alpha production preincubated for 30 mins followed by LPS stimulation measured after 20 to 24 hrs by enzyme immunoassay
Antagonist activity against EP4 in human whole blood assessed as reversal of PGE2-mediated suppression of LPS-induced TNF-alpha production preincubated for 30 mins followed by LPS stimulation measured after 20 to 24 hrs by enzyme immunoassayAntagonist activity against EP4 in human whole blood assessed as reversal of PGE2-mediated suppression of LPS-induced TNF-alpha production preincubated for 30 mins followed by LPS stimulation measured after 20 to 24 hrs by enzyme immunoassay
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Antagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Antagonist activity at rat EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 30 mins by HTRF assayAntagonist activity at rat EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 30 mins by HTRF assay
Antagonist activity at rat EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 30 mins by HTRF assayAntagonist activity at rat EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 30 mins by HTRF assay
cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.
Antagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Antagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Antagonist activity against EP4 in human whole blood assessed as reversal of PGE2-mediated suppression of LPS-induced TNF-alpha production preincubated for 30 mins followed by LPS stimulation measured after 20 to 24 hrs by enzyme immunoassayAntagonist activity against EP4 in human whole blood assessed as reversal of PGE2-mediated suppression of LPS-induced TNF-alpha production preincubated for 30 mins followed by LPS stimulation measured after 20 to 24 hrs by enzyme immunoassay
Antagonist activity at EP4 receptor in human whole blood assessed as reversal of inhibition of PGE2 mediated LPS-induced TNF alpha production pretreated for 30 mins using 3,3',5,5' tetramethylbiphenyl-4,4'-diamine substrate measured after 20 to 24 hrs by immunoassayAntagonist activity at EP4 receptor in human whole blood assessed as reversal of inhibition of PGE2 mediated LPS-induced TNF alpha production pretreated for 30 mins using 3,3',5,5' tetramethylbiphenyl-4,4'-diamine substrate measured after 20 to 24 hrs by immunoassay
Antagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serumDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serum
cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.
Antagonist activity at mouse EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at mouse EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assay
Antagonist activity against human EP4 expressed in HEK293 cells assessed as inhibition of PGE2-stimulated production of cAMP incubated for 20 mins by HTRF assayAntagonist activity against human EP4 expressed in HEK293 cells assessed as inhibition of PGE2-stimulated production of cAMP incubated for 20 mins by HTRF assay
Antagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha release pretreated for 30 mins followed by addition of PGE2 measured after 20 to 24 hrs by immunoassayAntagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha release pretreated for 30 mins followed by addition of PGE2 measured after 20 to 24 hrs by immunoassay
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Antagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISAAntagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISA
Antagonist activity at EP4 receptor in human whole blood assessed as blockade of inhibition of TNF-alpha-induced IP10 releaseAntagonist activity at EP4 receptor in human whole blood assessed as blockade of inhibition of TNF-alpha-induced IP10 release
Antagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serumDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serum
Antagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assay
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assay
Antagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Antagonist activity at recombinant human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP accumulation by scintillation proximity assay in presence of [125I]-cAMPAntagonist activity at recombinant human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP accumulation by scintillation proximity assay in presence of [125I]-cAMP
Antagonist activity at recombinant human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP accumulation by scintillation proximity assay in presence of [125I]-cAMPAntagonist activity at recombinant human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP accumulation by scintillation proximity assay in presence of [125I]-cAMP
Antagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha release pretreated for 30 mins followed by addition of PGE2 measured after 20 to 24 hrs by immunoassayAntagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha release pretreated for 30 mins followed by addition of PGE2 measured after 20 to 24 hrs by immunoassay
Inhibition of human EP4 transfected in human HEK293 cells assessed as reduction in PGE2-induced cAMP level incubated for 15 mins followed by PGE2 stimulation and measured every 2 mins for 30 mins by GloSensor cAMP AssayInhibition of human EP4 transfected in human HEK293 cells assessed as reduction in PGE2-induced cAMP level incubated for 15 mins followed by PGE2 stimulation and measured every 2 mins for 30 mins by GloSensor cAMP Assay
Displacement of [3H]-PGE2 from human EP4 receptor overexpressed in human ECV304 cell membranes by scintillation proximity assayDisplacement of [3H]-PGE2 from human EP4 receptor overexpressed in human ECV304 cell membranes by scintillation proximity assay
Displacement of [3H]PGE from human EP4 receptor expressed in HEK293 cell membranesDisplacement of [3H]PGE from human EP4 receptor expressed in HEK293 cell membranes
Antagonist activity at mouse EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at mouse EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serumDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serum
Antagonist activity against human EP4 expressed in HEK293 cells assessed as inhibition of PGE2-stimulated production of cAMP incubated for 20 mins by HTRF assayAntagonist activity against human EP4 expressed in HEK293 cells assessed as inhibition of PGE2-stimulated production of cAMP incubated for 20 mins by HTRF assay
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Antagonist activity at EP4 receptor in human whole blood assessed as reversal of inhibition of PGE2 mediated LPS-induced TNF alpha production pretreated for 30 mins using 3,3',5,5' tetramethylbiphenyl-4,4'-diamine substrate measured after 20 to 24 hrs by immunoassayAntagonist activity at EP4 receptor in human whole blood assessed as reversal of inhibition of PGE2 mediated LPS-induced TNF alpha production pretreated for 30 mins using 3,3',5,5' tetramethylbiphenyl-4,4'-diamine substrate measured after 20 to 24 hrs by immunoassay
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation countingDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation countingDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation countingDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assay
Antagonist activity against human EP4 expressed in HEK293 cells assessed as inhibition of PGE2-stimulated production of cAMP incubated for 20 mins by HTRF assayAntagonist activity against human EP4 expressed in HEK293 cells assessed as inhibition of PGE2-stimulated production of cAMP incubated for 20 mins by HTRF assay
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assay
Antagonist activity at human EP4 receptor in HEK293 cells by cell-based functional assayAntagonist activity at human EP4 receptor in HEK293 cells by cell-based functional assay
Displacement of [3H]PGE from human EP4 receptor expressed in HEK293 cell membranesDisplacement of [3H]PGE from human EP4 receptor expressed in HEK293 cell membranes
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation countingDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting
Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assay
Antagonist activity at human EP4 receptor in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulationAntagonist activity at human EP4 receptor in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation countingDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Antagonist activity at recombinant human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP accumulation by scintillation proximity assay in presence of [125I]-cAMPAntagonist activity at recombinant human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP accumulation by scintillation proximity assay in presence of [125I]-cAMP
Antagonist activity at recombinant human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP accumulation by scintillation proximity assay in presence of [125I]-cAMPAntagonist activity at recombinant human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP accumulation by scintillation proximity assay in presence of [125I]-cAMP
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay in presence of 10% human serumAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay in presence of 10% human serum
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by bead-based proximity assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by bead-based proximity assay
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Displacement of [3H]-PGE2 from human EP4 receptor overexpressed in human ECV304 cell membranes by scintillation proximity assayDisplacement of [3H]-PGE2 from human EP4 receptor overexpressed in human ECV304 cell membranes by scintillation proximity assay
Displacement of [3H]-PGE2 from recombinant human EP4 receptor expressed in HEK293 cell membranes after 90 mins by liquid scintillation counting analysisDisplacement of [3H]-PGE2 from recombinant human EP4 receptor expressed in HEK293 cell membranes after 90 mins by liquid scintillation counting analysis
Displacement of [3H]-PGE2 from recombinant human EP4 receptor expressed in HEK293 cell membranes after 90 mins by liquid scintillation counting analysisDisplacement of [3H]-PGE2 from recombinant human EP4 receptor expressed in HEK293 cell membranes after 90 mins by liquid scintillation counting analysis
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serumDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serum
cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.
Displacement of [3H]PGE from human EP4 receptor expressed in HEK293 cell membranesDisplacement of [3H]PGE from human EP4 receptor expressed in HEK293 cell membranes
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assay
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Antagonist activity at mouse EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at mouse EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serumDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serum
cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.
Antagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Antagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serumDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serum
cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serumDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serum
Antagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Displacement of [3H]-PGE2 from recombinant human EP4 receptor expressed in HEK293 cell membranes after 90 mins by liquid scintillation counting analysisDisplacement of [3H]-PGE2 from recombinant human EP4 receptor expressed in HEK293 cell membranes after 90 mins by liquid scintillation counting analysis
Displacement of [3H]-PGE2 from recombinant human EP4 receptor expressed in HEK293 cell membranes after 90 mins by liquid scintillation counting analysisDisplacement of [3H]-PGE2 from recombinant human EP4 receptor expressed in HEK293 cell membranes after 90 mins by liquid scintillation counting analysis
Antagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Antagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha release pretreated for 30 mins followed by addition of PGE2 measured after 20 to 24 hrs by immunoassayAntagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha release pretreated for 30 mins followed by addition of PGE2 measured after 20 to 24 hrs by immunoassay
Antagonist activity at EP4 receptor in human whole blood assessed as reversal of inhibition of PGE2 mediated LPS-induced TNF alpha production pretreated for 30 mins using 3,3',5,5' tetramethylbiphenyl-4,4'-diamine substrate measured after 20 to 24 hrs by immunoassayAntagonist activity at EP4 receptor in human whole blood assessed as reversal of inhibition of PGE2 mediated LPS-induced TNF alpha production pretreated for 30 mins using 3,3',5,5' tetramethylbiphenyl-4,4'-diamine substrate measured after 20 to 24 hrs by immunoassay
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assay
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assay
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Antagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Displacement of [3H]PGE2 from human prostanoid EP4 receptor expressed in cell membranes by scintillation countingDisplacement of [3H]PGE2 from human prostanoid EP4 receptor expressed in cell membranes by scintillation counting
Antagonist activity at mouse EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at mouse EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serumDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serum
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serumDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting in presence of 10% human serum
cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.
Displacement of [3H]PGE from human EP4 receptor expressed in HEK293 cell membranesDisplacement of [3H]PGE from human EP4 receptor expressed in HEK293 cell membranes
Antagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assayAntagonist activity at human EP4R assessed as inhibition of agonist-induced cAMP production by fluorescent cAMP tracer cAMP-d2 based FRET assay
Antagonist activity against EP4 in human whole blood assessed as reversal of PGE2-mediated suppression of LPS-induced TNF-alpha production preincubated for 30 mins followed by LPS stimulation measured after 20 to 24 hrs by enzyme immunoassayAntagonist activity against EP4 in human whole blood assessed as reversal of PGE2-mediated suppression of LPS-induced TNF-alpha production preincubated for 30 mins followed by LPS stimulation measured after 20 to 24 hrs by enzyme immunoassay
Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.Radioligand Binding: HEK-293 cells stably expressing the human or feline FP receptor, or EP1, EP2, EP3, or EP4 receptors were washed with TME buffer, scraped from the bottom of the flasks, and homogenized for 30 sec using a Brinkman PT 10/35 polytron. TME buffer was added to achieve a final 40 ml volume in the centrifuge tubes (the composition of TME is 100 mM TRIS base, 20 mM MgCl2, 2M EDTA; 10 N HCl is added to achieve a pH of 7.4).The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4 C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2 (5 nM) were performed in a 100 ul volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell.
cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.cAMPFunctional Assay: The assay is based on the competition between endogenous cAMP and exogenously added biotinylated cAMP. The capture of cAMP is achieved by using a specific antibody conjugated to Donor beads.Cell membranes prepared as described above, were resuspended in 1 ml stimulation buffer (HBSS 1x+BSA 0.1%+IBMX 0.5 mM+HEPES 5 mM+MgCl2 10 mM+GTP 1 nM+GDP 10 uM+ATP 100 uM - pH 7.4). Cell membranes were dispensed into white 384-well microplates at final concentration of 1 ug/well and used for the determination of cAMP with the alphascreen cAMP functional assay (EnVision-PerkinElmer). Cell membrane/anti-cAMP Acceptor beads mix (5 ul) and a mixture of analysed compounds (dissolved in 100% DMSO to a final maximal concentration of 0.01% DMSO)/PGE2 (5 ul) were incubated at room temperature (22-23° C.) for 30 min in the dark. The Biotinylated-cAMP and donor beads (15 ul) were dispensed into each well to start the competition reaction.
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Antagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISAAntagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISA
Antagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISAAntagonist activity at EP4 receptor in LPS-stimulated human whole blood assessed as inhibition of PGE2-induced TNF-alpha reduction preincubated for 30 mins followed by PGE2 addition measured after 24 hrs by ELISA
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP level by HTS assay
Antagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assayAntagonist activity at human EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-stimulated cAMP production after 1 hr by HTRF assay
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation countingDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation countingDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting
Antagonist activity at human EP4 receptor in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulationAntagonist activity at human EP4 receptor in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation
Antagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assayAntagonist activity at human EP4 expressed in CHO cells coexpressing G16-alpha assessed as intracellular calcium flux preincubated for 15 mins followed by addition of PGE2 by calcium flux assay
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation countingDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting
Antagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assayAntagonist activity at human prostanoid EP4 receptor expressed in HEK293 cells assessed as inhibition of PGE2-induced cAMP accumulation by scintillation proximity assay
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation countingDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation countingDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting
Displacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation countingDisplacement of [3H]PGE2 from human EP4 receptor expressed in HEK293 cells by scintillation counting
Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).