Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as pEC50 for CCL2-stimulated [35S]GTPgammaS binding at 1 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8.10 +/- 0.06 No_unit)Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as pEC50 for CCL2-stimulated [35S]GTPgammaS binding at 1 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8.10 +/- 0.06 No_unit)
Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as pEC50 for CCL2-stimulated [35S]GTPgammaS binding at 10 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8.10 +/- 0.06 No_unit)Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as pEC50 for CCL2-stimulated [35S]GTPgammaS binding at 10 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8.10 +/- 0.06 No_unit)
Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as EC50 for CCL2-stimulated [35S]GTPgammaS binding at 1 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8 nM)Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as EC50 for CCL2-stimulated [35S]GTPgammaS binding at 1 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8 nM)
Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as EC50 for CCL2-stimulated [35S]GTPgammaS binding at 10 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8 nM)Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as EC50 for CCL2-stimulated [35S]GTPgammaS binding at 10 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8 nM)
Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as pEC50 for CCL2-stimulated [35S]GTPgammaS binding at 10 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8.10 +/- 0.06 No_unit)Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as pEC50 for CCL2-stimulated [35S]GTPgammaS binding at 10 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8.10 +/- 0.06 No_unit)
Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as EC50 for CCL2-stimulated [35S]GTPgammaS binding at 10 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8 nM)Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as EC50 for CCL2-stimulated [35S]GTPgammaS binding at 10 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8 nM)
Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as pEC50 for CCL2-stimulated [35S]GTPgammaS binding at 30 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8.10 +/- 0.06 No_unit)Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as pEC50 for CCL2-stimulated [35S]GTPgammaS binding at 30 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8.10 +/- 0.06 No_unit)
Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as EC50 for CCL2-stimulated [35S]GTPgammaS binding at 30 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8 nM)Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as EC50 for CCL2-stimulated [35S]GTPgammaS binding at 30 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8 nM)
Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as pEC50 for CCL2-stimulated [35S]GTPgammaS binding at 3 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8.10 +/- 0.06 No_unit)Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as pEC50 for CCL2-stimulated [35S]GTPgammaS binding at 3 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8.10 +/- 0.06 No_unit)
Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as EC50 for CCL2-stimulated [35S]GTPgammaS binding at 3 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8 nM)Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as EC50 for CCL2-stimulated [35S]GTPgammaS binding at 3 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8 nM)
Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as pEC50 for CCL2-stimulated [35S]GTPgammaS binding at 100 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8.10 +/- 0.06 No_unit)Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as pEC50 for CCL2-stimulated [35S]GTPgammaS binding at 100 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8.10 +/- 0.06 No_unit)
Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as EC50 for CCL2-stimulated [35S]GTPgammaS binding at 100 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8 nM)Non-competitive insurmountable antagonist activity at human CCR2 expressed in human U2OS cell membranes assessed as EC50 for CCL2-stimulated [35S]GTPgammaS binding at 100 nM preincubated for 30 mins followed by [35S]GTPgammaS addition and measured after 90 mins (Rvb = 8 nM)
Antagonist activity at human CCR2 receptor expressed in CHO cells by chemotaxis assayAntagonist activity at human CCR2 receptor expressed in CHO cells by chemotaxis assay
Antagonist activity at human C-C chemokine receptor type 2 expressed in THP-1 cells assessed as inhibition of CCL2-induced calcium flux preincubated for 10 mins followed by CCL2 addition by Fluo-4 dye based FLIPR assayAntagonist activity at human C-C chemokine receptor type 2 expressed in THP-1 cells assessed as inhibition of CCL2-induced calcium flux preincubated for 10 mins followed by CCL2 addition by Fluo-4 dye based FLIPR assay
Antagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxis
Antagonist activity at CCR2 in human monocytes assessed as reduction of chemotaxis in presence of 0.1 M BSAAntagonist activity at CCR2 in human monocytes assessed as reduction of chemotaxis in presence of 0.1 M BSA
Antagonist activity at CCR2 in human monocytes assessed as reduction of chemotaxis in presence of 0.1 M BSAAntagonist activity at CCR2 in human monocytes assessed as reduction of chemotaxis in presence of 0.1 M BSA
Antagonist activity at human CCR2 receptor expressed in CHO cells by chemotaxis assayAntagonist activity at human CCR2 receptor expressed in CHO cells by chemotaxis assay
Antagonist activity against CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis at 37 degCAntagonist activity against CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis at 37 degC
Antagonist activity at CCR2 in human PBMC assessed as MCP1-induced calcium flux by fluorescence-imaging plate reader assayAntagonist activity at CCR2 in human PBMC assessed as MCP1-induced calcium flux by fluorescence-imaging plate reader assay
Antagonist activity at CCR2 in human THP1 cells assessed as reduction in MCP1-induced chemotaxis measured after 30 mins by calcein-AM dye based fluorescence assayAntagonist activity at CCR2 in human THP1 cells assessed as reduction in MCP1-induced chemotaxis measured after 30 mins by calcein-AM dye based fluorescence assay
Antagonist activity at human CCR2 receptor expressed in CHO cells by chemotaxis assayAntagonist activity at human CCR2 receptor expressed in CHO cells by chemotaxis assay
Displacement of [125I]MCP1 from CCR2 in human PBMC by millipore filter plate assayDisplacement of [125I]MCP1 from CCR2 in human PBMC by millipore filter plate assay
Inhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperatureInhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperature
Antagonist activity against CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis at 37 degCAntagonist activity against CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis at 37 degC
Antagonist activity at CCR2 in human PBMC assessed as MCP1-induced calcium flux by fluorescence-imaging plate reader assayAntagonist activity at CCR2 in human PBMC assessed as MCP1-induced calcium flux by fluorescence-imaging plate reader assay
Antagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis after 45 mins in presence of 0.1 M bovine serum albuminAntagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis after 45 mins in presence of 0.1 M bovine serum albumin
Antagonist activity at CCR2 in human monocytes assessed as reduction of chemotaxis in presence of 0.1 M BSAAntagonist activity at CCR2 in human monocytes assessed as reduction of chemotaxis in presence of 0.1 M BSA
Antagonist activity at human CCR2 in PBMCs assessed as inhibition of chemotaxisAntagonist activity at human CCR2 in PBMCs assessed as inhibition of chemotaxis
Inhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperatureInhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperature
Antagonist activity against CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis at 37 degCAntagonist activity against CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis at 37 degC
Antagonist activity at CCR2 in human THP1 cells assessed as reduction in MCP1-induced chemotaxis measured after 30 mins by calcein-AM dye based fluorescence assayAntagonist activity at CCR2 in human THP1 cells assessed as reduction in MCP1-induced chemotaxis measured after 30 mins by calcein-AM dye based fluorescence assay
Antagonist activity at CCR2 in human monocytes assessed as reduction of chemotaxis in presence of 0.1 M BSAAntagonist activity at CCR2 in human monocytes assessed as reduction of chemotaxis in presence of 0.1 M BSA
Inhibition of MCP1-stimulated chemotaxis in monocytes transfected with human CCR2Inhibition of MCP1-stimulated chemotaxis in monocytes transfected with human CCR2
Antagonist activity at human C-C chemokine receptor type 2 expressed in THP-1 cells assessed as inhibition of CCL2-induced calcium flux preincubated for 10 mins followed by CCL2 addition by Fluo-4 dye based FLIPR assayAntagonist activity at human C-C chemokine receptor type 2 expressed in THP-1 cells assessed as inhibition of CCL2-induced calcium flux preincubated for 10 mins followed by CCL2 addition by Fluo-4 dye based FLIPR assay
Inhibition of CCR2 in human THP1 cells assessed as reduction in MCP1-induced chemotaxis measured after 10 mins by calcein-AM dye based fluorescence assayInhibition of CCR2 in human THP1 cells assessed as reduction in MCP1-induced chemotaxis measured after 10 mins by calcein-AM dye based fluorescence assay
Antagonist activity at CCR2 in human PBMC assessed as MCP1-induced calcium flux by fluorescence-imaging plate reader assayAntagonist activity at CCR2 in human PBMC assessed as MCP1-induced calcium flux by fluorescence-imaging plate reader assay
Antagonist activity at CCR2 in human THP1 cells assessed as reduction in MCP1-induced chemotaxis measured after 30 mins by calcein-AM dye based fluorescence assayAntagonist activity at CCR2 in human THP1 cells assessed as reduction in MCP1-induced chemotaxis measured after 30 mins by calcein-AM dye based fluorescence assay
Inhibition of MCP1-stimulated chemotaxis in monocytes transfected with human CCR2Inhibition of MCP1-stimulated chemotaxis in monocytes transfected with human CCR2
Inhibition of MCP1-stimulated chemotaxis in monocytes transfected with human CCR2Inhibition of MCP1-stimulated chemotaxis in monocytes transfected with human CCR2
Inhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperatureInhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperature
Antagonist activity at CCR2 in human THP1 cells assessed as reduction in MCP1-induced chemotaxis measured after 30 mins by calcein-AM dye based fluorescence assayAntagonist activity at CCR2 in human THP1 cells assessed as reduction in MCP1-induced chemotaxis measured after 30 mins by calcein-AM dye based fluorescence assay
Antagonist activity at human CCR2 receptor expressed in CHO cells by chemotaxis assayAntagonist activity at human CCR2 receptor expressed in CHO cells by chemotaxis assay
Inhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperatureInhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperature
Inhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperatureInhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperature
Antagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxis
Antagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxis
Antagonist activity against human CCR2B expressed in Chem-1 cells assessed as inhibition of human CCL2-induced intracellular calcium flux incubated for 10 mins by Fluo-4 AM dye based intracellular calcium flux assayAntagonist activity against human CCR2B expressed in Chem-1 cells assessed as inhibition of human CCL2-induced intracellular calcium flux incubated for 10 mins by Fluo-4 AM dye based intracellular calcium flux assay
Antagonist activity at human CCR2b expressed in human Chem1 cells assessed as inhibition of human recombinant CCL2 induced Ca2+ mobilization preincubated for 10 mins followed by CCL2 induction measured every sec for 120 secs by Fluo-4-AM dye-based fluorescence assayAntagonist activity at human CCR2b expressed in human Chem1 cells assessed as inhibition of human recombinant CCL2 induced Ca2+ mobilization preincubated for 10 mins followed by CCL2 induction measured every sec for 120 secs by Fluo-4-AM dye-based fluorescence assay
Displacement of radiolabeled MCP1 from CCR2 in human PBMC by millipore filter plate assayDisplacement of radiolabeled MCP1 from CCR2 in human PBMC by millipore filter plate assay
Antagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxis
Antagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis after 30 minsAntagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis after 30 mins
Antagonist activity at human CCR2 in PBMCs assessed as inhibition of chemotaxisAntagonist activity at human CCR2 in PBMCs assessed as inhibition of chemotaxis
Antagonist activity at human CCR2 receptor expressed in CHO cells by chemotaxis assayAntagonist activity at human CCR2 receptor expressed in CHO cells by chemotaxis assay
Antagonist activity at human CCR2 receptor expressed in CHO cells by chemotaxis assayAntagonist activity at human CCR2 receptor expressed in CHO cells by chemotaxis assay
Antagonist activity at human CCR2 receptor expressed in CHO cells by chemotaxis assayAntagonist activity at human CCR2 receptor expressed in CHO cells by chemotaxis assay
Displacement of [125I]MCP1 from CCR2 in human PBMC after 30 mins by gamma counterDisplacement of [125I]MCP1 from CCR2 in human PBMC after 30 mins by gamma counter
Inhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperatureInhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperature
Antagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxis
Antagonist activity at human C-C chemokine receptor type 2 expressed in THP-1 cells assessed as inhibition of CCL2-induced calcium flux preincubated for 10 mins followed by CCL2 addition by Fluo-4 dye based FLIPR assayAntagonist activity at human C-C chemokine receptor type 2 expressed in THP-1 cells assessed as inhibition of CCL2-induced calcium flux preincubated for 10 mins followed by CCL2 addition by Fluo-4 dye based FLIPR assay
Displacement of [125I]MCP1 from CCR2 in human PBMC measured after 30 mins by Microscintillation counting analysisDisplacement of [125I]MCP1 from CCR2 in human PBMC measured after 30 mins by Microscintillation counting analysis
Inhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperatureInhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperature
Antagonist activity at human C-C chemokine receptor type 2 expressed in THP-1 cells assessed as inhibition of CCL2-induced calcium flux preincubated for 10 mins followed by CCL2 addition by Fluo-4 dye based FLIPR assayAntagonist activity at human C-C chemokine receptor type 2 expressed in THP-1 cells assessed as inhibition of CCL2-induced calcium flux preincubated for 10 mins followed by CCL2 addition by Fluo-4 dye based FLIPR assay
Antagonist activity against CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis at 37 degCAntagonist activity against CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis at 37 degC
Antagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis after 45 mins in presence of 0.1 M bovine serum albuminAntagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis after 45 mins in presence of 0.1 M bovine serum albumin
Antagonist activity at rat C-C chemokine receptor type 2 assessed as inhibition of CCL2-induced calcium fluxAntagonist activity at rat C-C chemokine receptor type 2 assessed as inhibition of CCL2-induced calcium flux
Displacement of [125I]MCP1 from CCR2 in human PBMC by millipore filter plate assayDisplacement of [125I]MCP1 from CCR2 in human PBMC by millipore filter plate assay
Displacement of [125I]MCP1 from CCR2 in human PBMC by millipore filter plate assayDisplacement of [125I]MCP1 from CCR2 in human PBMC by millipore filter plate assay
Displacement of [125I]MCP1 from CCR2 in human PBMC measured after 30 mins by Microscintillation counting analysisDisplacement of [125I]MCP1 from CCR2 in human PBMC measured after 30 mins by Microscintillation counting analysis
Displacement of [125]hMCP1 from human CCR2 receptor expressed in human monocytesDisplacement of [125]hMCP1 from human CCR2 receptor expressed in human monocytes
Antagonist activity at human CCR2 assessed as inhibition of MCP1 induced chemotaxis after 30 minsAntagonist activity at human CCR2 assessed as inhibition of MCP1 induced chemotaxis after 30 mins
Displacement of [125I]MCP1 from CCR2 in human PBMC by millipore filter plate assayDisplacement of [125I]MCP1 from CCR2 in human PBMC by millipore filter plate assay
Displacement of [125]hMCP1 from human CCR2 receptor expressed in human monocytesDisplacement of [125]hMCP1 from human CCR2 receptor expressed in human monocytes
Antagonist activity at CCR2 in human PBMC assessed as MCP1-induced calcium flux by fluorescence-imaging plate reader assayAntagonist activity at CCR2 in human PBMC assessed as MCP1-induced calcium flux by fluorescence-imaging plate reader assay
Displacement of [3H]-CCR2-RA-[R] from wild type human CCR2 expressed in CHO cell membranes incubated for 2 hrs by scintillation counting methodDisplacement of [3H]-CCR2-RA-[R] from wild type human CCR2 expressed in CHO cell membranes incubated for 2 hrs by scintillation counting method
Antagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis after 45 mins in presence of 0.5 M bovine serum albuminAntagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis after 45 mins in presence of 0.5 M bovine serum albumin
Antagonist activity at CCR2 in human THP1 cells assessed as reduction in MCP1-induced chemotaxis measured after 30 mins by calcein-AM dye based fluorescence assayAntagonist activity at CCR2 in human THP1 cells assessed as reduction in MCP1-induced chemotaxis measured after 30 mins by calcein-AM dye based fluorescence assay
Antagonist activity at CCR2 in human THP1 cells assessed as reduction in MCP1-induced chemotaxis measured after 30 mins by calcein-AM dye based fluorescence assayAntagonist activity at CCR2 in human THP1 cells assessed as reduction in MCP1-induced chemotaxis measured after 30 mins by calcein-AM dye based fluorescence assay
Antagonist activity at human C-C chemokine receptor type 2 expressed in THP-1 cells assessed as inhibition of CCL2-induced calcium flux preincubated for 10 mins followed by CCL2 addition by Fluo-4 dye based FLIPR assayAntagonist activity at human C-C chemokine receptor type 2 expressed in THP-1 cells assessed as inhibition of CCL2-induced calcium flux preincubated for 10 mins followed by CCL2 addition by Fluo-4 dye based FLIPR assay
Antagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium levelAntagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium level
Inhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperatureInhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperature
Antagonist activity against CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis at 37 degCAntagonist activity against CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis at 37 degC
Antagonist activity at CCR2 receptor in human PBMC assessed as inhibition of MCP1-mediated leukocyte chemotaxis after 30 mins by microscopyAntagonist activity at CCR2 receptor in human PBMC assessed as inhibition of MCP1-mediated leukocyte chemotaxis after 30 mins by microscopy
Antagonist activity at human CCR2 assessed as inhibition of MCP1 induced chemotaxis after 30 minsAntagonist activity at human CCR2 assessed as inhibition of MCP1 induced chemotaxis after 30 mins
Displacement of radiolabeled MCP1 from CCR2 in human PBMC by millipore filter plate assayDisplacement of radiolabeled MCP1 from CCR2 in human PBMC by millipore filter plate assay
Inhibition of MCP1-stimulated chemotaxis in monocytes transfected with human CCR2Inhibition of MCP1-stimulated chemotaxis in monocytes transfected with human CCR2
Antagonist activity at CCR2 receptor in human PBMC assessed as inhibition of MCP1-mediated leukocyte chemotaxis after 30 mins by microscopyAntagonist activity at CCR2 receptor in human PBMC assessed as inhibition of MCP1-mediated leukocyte chemotaxis after 30 mins by microscopy
Displacement of [125I]MCP1 from human CCR2 I40L mutant expressed in CHO cellsDisplacement of [125I]MCP1 from human CCR2 I40L mutant expressed in CHO cells
Antagonist activity against human CCR2 in human THP1 cells using [I125]MCP-1 by scintillation countingAntagonist activity against human CCR2 in human THP1 cells using [I125]MCP-1 by scintillation counting
Antagonist activity at CCR2 by monocyte transmigration assay in pesence of 0.5% BSAAntagonist activity at CCR2 by monocyte transmigration assay in pesence of 0.5% BSA
Antagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis
Antagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 1 hr by crystal violet stainingAntagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 1 hr by crystal violet staining
Antagonist activity at human CCR2 expressed in HEK293 cells assessed as inhibition of beta-arrestin recruitment pre-incubated for 20 mins followed by drug wash out before CCL2 stimulation and followed by further incubation for 90 mins by NanoBiT CCR2 assay basedAntagonist activity at human CCR2 expressed in HEK293 cells assessed as inhibition of beta-arrestin recruitment pre-incubated for 20 mins followed by drug wash out before CCL2 stimulation and followed by further incubation for 90 mins by NanoBiT CCR2 assay based
Antagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Antagonist activity at human TEV cleavage site-linked CCR2b expressed in human U2OS cells harboring beta-lactamase reporter gene assessed as inhibition of CCL2-induced beta-arrestin recruitment incubated for 30 mins followed by CCL2 stimulation and measured after 16 hrs by Tango assayAntagonist activity at human TEV cleavage site-linked CCR2b expressed in human U2OS cells harboring beta-lactamase reporter gene assessed as inhibition of CCL2-induced beta-arrestin recruitment incubated for 30 mins followed by CCL2 stimulation and measured after 16 hrs by Tango assay
Displacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assayDisplacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assay
Displacement of [125I]-MCP1 from CCR2 in human THP1 cells after 2 hrs by microplate scintillation and luminescence countingDisplacement of [125I]-MCP1 from CCR2 in human THP1 cells after 2 hrs by microplate scintillation and luminescence counting
Displacement of [125I]MCP1 from human CCR2 I40L mutant expressed in CHO cellsDisplacement of [125I]MCP1 from human CCR2 I40L mutant expressed in CHO cells
Displacement of [125I]MCP1 from human CCR2 in PBMC after 30 mins by gamma countingDisplacement of [125I]MCP1 from human CCR2 in PBMC after 30 mins by gamma counting
Displacement of [125I]MCP1 from human CCR2 in PBMC after 30 mins by gamma countingDisplacement of [125I]MCP1 from human CCR2 in PBMC after 30 mins by gamma counting
Displacement of [125I]MCP1 from CCR2 in human PBMC after 30 mins by gamma counterDisplacement of [125I]MCP1 from CCR2 in human PBMC after 30 mins by gamma counter
Antagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxis
Antagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis after 30 minsAntagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis after 30 mins
Displacement of [125I]MCP1 from CCR2 in human PBMC after 30 mins by gamma counterDisplacement of [125I]MCP1 from CCR2 in human PBMC after 30 mins by gamma counter
Antagonist activity at CCR2 in human PBMCs assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMCs assessed as inhibition of MCP1-induced chemotaxis
Antagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 1 hr by crystal violet stainingAntagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 1 hr by crystal violet staining
Antagonist activity at CCR2 receptor in PBMC assessed as inhibition of MCP1-induced calcium mobilizationAntagonist activity at CCR2 receptor in PBMC assessed as inhibition of MCP1-induced calcium mobilization
Antagonist activity at CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced calcium mobilizationAntagonist activity at CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced calcium mobilization
Antagonist activity at human CCR2 expressed in HEK293 cells assessed as inhibition of beta-arrestin recruitment pre-incubated for 20 mins followed by drug wash out before CCL2 stimulation and followed by further incubation for 90 mins by NanoBiT CCR2 assay basedAntagonist activity at human CCR2 expressed in HEK293 cells assessed as inhibition of beta-arrestin recruitment pre-incubated for 20 mins followed by drug wash out before CCL2 stimulation and followed by further incubation for 90 mins by NanoBiT CCR2 assay based
Displacement of [125I]-MCP-1 from CCR2 in human THP-1 cells after 2 hrs by microplate scintillation and luminescence counter analysisDisplacement of [125I]-MCP-1 from CCR2 in human THP-1 cells after 2 hrs by microplate scintillation and luminescence counter analysis
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Antagonist activity at CCR2 in human THP1 cells assessed as inhibition of CCL2-induced chemotaxisAntagonist activity at CCR2 in human THP1 cells assessed as inhibition of CCL2-induced chemotaxis
Displacement of [125I]-MCP1 from CCR2 in human THP1 cells after 2 hrs by microplate scintillation and luminescence countingDisplacement of [125I]-MCP1 from CCR2 in human THP1 cells after 2 hrs by microplate scintillation and luminescence counting
Displacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cellsDisplacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cells
Inhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assayInhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assay
Inhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assayInhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assay
Inhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assayInhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assay
Inhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assayInhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assay
Inhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assayInhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assay
Inhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assayInhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assay
Inhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assayInhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assay
Inhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assayInhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assay
Inhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assayInhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assay
Antagonist activity at CCR2 in rat spleen cells assessed as inhibition of CCL2-induced chemotaxisAntagonist activity at CCR2 in rat spleen cells assessed as inhibition of CCL2-induced chemotaxis
Antagonist activity at CCR2 in rat spleen cells assessed as inhibition of CCL2-induced chemotaxisAntagonist activity at CCR2 in rat spleen cells assessed as inhibition of CCL2-induced chemotaxis
Antagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxis
Compound concentration that causes 50 % inhibition of binding of specific radioligand to human CCR2 receptorCompound concentration that causes 50 % inhibition of binding of specific radioligand to human CCR2 receptor
Displacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation countingDisplacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation counting
Inhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assayInhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assay
Inhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assayInhibition of MCP1-induced cell migration in U937 cells expressing CCR2 by chemotaxis assay
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Antagonist activity at human CCR2 expressed in HEK293 cells assessed as inhibition of beta-arrestin recruitment pre-incubated for 20 mins followed by drug wash out before CCL2 stimulation and followed by further incubation for 90 mins by NanoBiT CCR2 assay basedAntagonist activity at human CCR2 expressed in HEK293 cells assessed as inhibition of beta-arrestin recruitment pre-incubated for 20 mins followed by drug wash out before CCL2 stimulation and followed by further incubation for 90 mins by NanoBiT CCR2 assay based
Antagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxis
Displacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assayDisplacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assay
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Displacement of 125I-hMCP1 from CCR2 expressed in human peripheral blood monocytes after 60 minsDisplacement of 125I-hMCP1 from CCR2 expressed in human peripheral blood monocytes after 60 mins
Displacement of 125I-hMCP1 from CCR2 expressed in human peripheral blood monocytes after 60 minsDisplacement of 125I-hMCP1 from CCR2 expressed in human peripheral blood monocytes after 60 mins
Antagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxis
Antagonist activity at CCR2 in human PBMCs assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMCs assessed as inhibition of MCP1-induced chemotaxis
Antagonist activity at human CCR2 expressed in THP1 cells assessed as inhibition of MCP-1 induced chemotaxisAntagonist activity at human CCR2 expressed in THP1 cells assessed as inhibition of MCP-1 induced chemotaxis
Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).
Antagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Antagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxis
Antagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxis
Antagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Antagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Antagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Antagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium levelAntagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium level
Binding Assay: THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30° C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 μL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.). Blank values (buffer only) were subtracted from all values and drug treated values were compared to vehicle treated values. 1 μM cold MCP-1 was used for nonspecific binding.Binding Assay: THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30° C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 μL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.). Blank values (buffer only) were subtracted from all values and drug treated values were compared to vehicle treated values. 1 μM cold MCP-1 was used for nonspecific binding.
Displacement of [125I]MCP-1 from CCR2 in human PBMC by radioligand displacement assayDisplacement of [125I]MCP-1 from CCR2 in human PBMC by radioligand displacement assay
Displacement of [125I]MCP1 from human CCR2 in PBMC after 30 mins by gamma countingDisplacement of [125I]MCP1 from human CCR2 in PBMC after 30 mins by gamma counting
Displacement of [125I]MCP1 from human CCR2 in PBMC after 30 mins by gamma countingDisplacement of [125I]MCP1 from human CCR2 in PBMC after 30 mins by gamma counting
Displacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assay
Displacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assay
Displacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assay
Displacement of [125I]MCP1 from human CCR2 preincubated 1 hrs by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated 1 hrs by human whole cell binding assay
Antagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxis
Antagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium levelAntagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium level
Displacement of [125I]-MCP1 from CCR2 in human THP1 cells after 2 hrs by microplate scintillation and luminescence countingDisplacement of [125I]-MCP1 from CCR2 in human THP1 cells after 2 hrs by microplate scintillation and luminescence counting
Antagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium levelAntagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium level
Concentration required to inhibit [125I]MCP-1 binding to Chemokine receptor 2BConcentration required to inhibit [125I]MCP-1 binding to Chemokine receptor 2B
Concentration required to inhibit [125I]MCP-1 binding to Chemokine receptor 2BConcentration required to inhibit [125I]MCP-1 binding to Chemokine receptor 2B
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Displacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cellsDisplacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cells
Antagonist activity against mouse CCR2B expressed in human U2OS cells co-expressing beta-arrestin assessed as inhibition of mouse CCL2-induced beta-arrestin recruitment pre-incubated for 10 mins before mouse CCL2 stimulation for 90 mins by luminescence based assayAntagonist activity against mouse CCR2B expressed in human U2OS cells co-expressing beta-arrestin assessed as inhibition of mouse CCL2-induced beta-arrestin recruitment pre-incubated for 10 mins before mouse CCL2 stimulation for 90 mins by luminescence based assay
Displacement of 125I-hMCP1 from CCR2 expressed in human peripheral blood monocytes after 60 minsDisplacement of 125I-hMCP1 from CCR2 expressed in human peripheral blood monocytes after 60 mins
Displacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cellsDisplacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cells
Displacement of [125I]MCP-1 from CCR2 in human PBMC by radioligand displacement assayDisplacement of [125I]MCP-1 from CCR2 in human PBMC by radioligand displacement assay
Antagonist activity against human CCR2 receptor assessed as inhibition of MCP1-induced migration of human peripheral blood monocytesAntagonist activity against human CCR2 receptor assessed as inhibition of MCP1-induced migration of human peripheral blood monocytes
Displacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cellsDisplacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cells
Displacement of radiolabeled MCP1 from CCR2 in human PBMC by millipore filter plate assayDisplacement of radiolabeled MCP1 from CCR2 in human PBMC by millipore filter plate assay
Displacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assayDisplacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assay
Antagonist activity at CCR2 (unknown origin) expressed in CHOK1 cells co-expressing Ga16 assessed as inhibition of CCL2 induced intracellular Ca2+ mobilization preincubated for 10 mins followed by CCL2 addition by fluo-4 AM dye based assayAntagonist activity at CCR2 (unknown origin) expressed in CHOK1 cells co-expressing Ga16 assessed as inhibition of CCL2 induced intracellular Ca2+ mobilization preincubated for 10 mins followed by CCL2 addition by fluo-4 AM dye based assay
Antagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis
Antagonist activity at CCR2 receptor in human whole blood assessed as inhibition of alexa-tagged MCP-induced effect 30 mins by flow cytometryAntagonist activity at CCR2 receptor in human whole blood assessed as inhibition of alexa-tagged MCP-induced effect 30 mins by flow cytometry
Antagonist activity at human CCR2 assessed as inhibition of MCP1 induced chemotaxis after 30 minsAntagonist activity at human CCR2 assessed as inhibition of MCP1 induced chemotaxis after 30 mins
Antagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxis
Antagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Antagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium levelAntagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium level
Displacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation countingDisplacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation counting
Displacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation countingDisplacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation counting
Displacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation countingDisplacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation counting
Displacement of [125I]MCP-1 from human CCR2b expressed in human monocyte cellsDisplacement of [125I]MCP-1 from human CCR2b expressed in human monocyte cells
Displacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assay
Displacement of [3H]-CCR2-RA-[R] from wild type human CCR2 expressed in CHO cell membranes incubated for 2 hrs by scintillation counting methodDisplacement of [3H]-CCR2-RA-[R] from wild type human CCR2 expressed in CHO cell membranes incubated for 2 hrs by scintillation counting method
Displacement of [3H]-CCR2-RA-[R] from wild type human CCR2 expressed in CHO cell membranes incubated for 2 hrs by scintillation counting methodDisplacement of [3H]-CCR2-RA-[R] from wild type human CCR2 expressed in CHO cell membranes incubated for 2 hrs by scintillation counting method
Displacement of [125I]MCP1 from human CCR2 preincubated 1 hrs by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated 1 hrs by human whole cell binding assay
Antagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxis
Displacement of [125I]MCP1 from CCR2 in human PBMC by millipore filter plate assayDisplacement of [125I]MCP1 from CCR2 in human PBMC by millipore filter plate assay
Displacement of [3H]-CCR2-RA-[R] from wild type human CCR2 expressed in CHO cell membranes incubated for 2 hrs by scintillation counting methodDisplacement of [3H]-CCR2-RA-[R] from wild type human CCR2 expressed in CHO cell membranes incubated for 2 hrs by scintillation counting method
Displacement of [3H]-CCR2-RA-[R] from wild type human CCR2 expressed in CHO cell membranes incubated for 2 hrs by scintillation counting methodDisplacement of [3H]-CCR2-RA-[R] from wild type human CCR2 expressed in CHO cell membranes incubated for 2 hrs by scintillation counting method
Antagonist activity at CCR2 in human PBMCs assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMCs assessed as inhibition of MCP1-induced chemotaxis
Antagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxis
Displacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assay
Antagonist activity at mouse CCR2 assessed as inhibition of MCP1 induced chemotaxis after 30 minsAntagonist activity at mouse CCR2 assessed as inhibition of MCP1 induced chemotaxis after 30 mins
Antagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium levelAntagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium level
Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).
Binding Assay: THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30° C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 μL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.). Blank values (buffer only) were subtracted from all values and drug treated values were compared to vehicle treated values. 1 μM cold MCP-1 was used for nonspecific binding.Binding Assay: THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30° C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 μL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.). Blank values (buffer only) were subtracted from all values and drug treated values were compared to vehicle treated values. 1 μM cold MCP-1 was used for nonspecific binding.
Displacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation countingDisplacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation counting
Antagonist activity at human CCR2 in PBMCs assessed as inhibition of chemotaxisAntagonist activity at human CCR2 in PBMCs assessed as inhibition of chemotaxis
Antagonist activity at mouse CCR2 receptor expressed in human THP-1 cells assessed as inhibition of MCP1-induced Ca2+ flux by FLIPR analysisAntagonist activity at mouse CCR2 receptor expressed in human THP-1 cells assessed as inhibition of MCP1-induced Ca2+ flux by FLIPR analysis
Concentration required to inhibit [125I]MCP-1 binding to Chemokine receptor 2BConcentration required to inhibit [125I]MCP-1 binding to Chemokine receptor 2B
Displacement of [125I]-MCP1 from CCR2 in human THP1 cells after 2 hrs by microplate scintillation and luminescence countingDisplacement of [125I]-MCP1 from CCR2 in human THP1 cells after 2 hrs by microplate scintillation and luminescence counting
Displacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cellsDisplacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cells
Antagonist activity against human CCR2B expressed in Chem-1 cells assessed as inhibition of human CCL2-induced intracellular calcium flux incubated for 10 mins by Fluo-4 AM dye based intracellular calcium flux assayAntagonist activity against human CCR2B expressed in Chem-1 cells assessed as inhibition of human CCL2-induced intracellular calcium flux incubated for 10 mins by Fluo-4 AM dye based intracellular calcium flux assay
Antagonist activity at CCR2 in mouse spleen cells assessed as inhibition of CCL2-induced chemotaxisAntagonist activity at CCR2 in mouse spleen cells assessed as inhibition of CCL2-induced chemotaxis
Antagonist activity at CCR2 in mouse spleen cells assessed as inhibition of CCL2-induced chemotaxisAntagonist activity at CCR2 in mouse spleen cells assessed as inhibition of CCL2-induced chemotaxis
Antagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxis
Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).
Displacement of 125I-hMCP1 from CCR2 expressed in human peripheral blood monocytes after 60 minsDisplacement of 125I-hMCP1 from CCR2 expressed in human peripheral blood monocytes after 60 mins
Displacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assayDisplacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assay
Displacement of radiolabeled MCP1 from CCR2 in human PBMC by millipore filter plate assayDisplacement of radiolabeled MCP1 from CCR2 in human PBMC by millipore filter plate assay
Antagonist activity at CCR2 (unknown origin) expressed in CHOK1 cells co-expressing Ga16 assessed as inhibition of CCL2 induced intracellular Ca2+ mobilization preincubated for 10 mins followed by CCL2 addition by fluo-4 AM dye based assayAntagonist activity at CCR2 (unknown origin) expressed in CHOK1 cells co-expressing Ga16 assessed as inhibition of CCL2 induced intracellular Ca2+ mobilization preincubated for 10 mins followed by CCL2 addition by fluo-4 AM dye based assay
Antagonist activity against human CCR2B expressed in Chem-1 cells assessed as inhibition of human CCL2-induced intracellular calcium flux incubated for 10 mins by Fluo-4 AM dye based intracellular calcium flux assayAntagonist activity against human CCR2B expressed in Chem-1 cells assessed as inhibition of human CCL2-induced intracellular calcium flux incubated for 10 mins by Fluo-4 AM dye based intracellular calcium flux assay
Displacement of [125I]MCP1 from human CCR2 in PBMC after 30 mins by gamma countingDisplacement of [125I]MCP1 from human CCR2 in PBMC after 30 mins by gamma counting
Antagonist activity at CCR2 (unknown origin) expressed in CHOK1 cells co-expressing Ga16 assessed as inhibition of CCL2 induced intracellular Ca2+ mobilization preincubated for 10 mins followed by CCL2 addition by fluo-4 AM dye based assayAntagonist activity at CCR2 (unknown origin) expressed in CHOK1 cells co-expressing Ga16 assessed as inhibition of CCL2 induced intracellular Ca2+ mobilization preincubated for 10 mins followed by CCL2 addition by fluo-4 AM dye based assay
Antagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxis
Displacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assayDisplacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assay
Displacement of [125I]MCP-1 from human CCR2b expressed in human monocyte cellsDisplacement of [125I]MCP-1 from human CCR2b expressed in human monocyte cells
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Antagonist activity against CCR2 in human THP-1 cells assessed as inhibition of MCP-1 induced chemotaxis after 3 hrsAntagonist activity against CCR2 in human THP-1 cells assessed as inhibition of MCP-1 induced chemotaxis after 3 hrs
Antagonist activity at CCR2 in human PBMC assessed as MCP1-induced calcium flux by fluorescence-imaging plate assayAntagonist activity at CCR2 in human PBMC assessed as MCP1-induced calcium flux by fluorescence-imaging plate assay
Antagonist activity at CCR2 in human PBMC assessed as MCP1-induced calcium flux by fluorescence-imaging plate assayAntagonist activity at CCR2 in human PBMC assessed as MCP1-induced calcium flux by fluorescence-imaging plate assay
Antagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis
Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).
Displacement of [125I]-MCP1 from CCR2 in human THP1 cells after 2 hrs by microplate scintillation and luminescence countingDisplacement of [125I]-MCP1 from CCR2 in human THP1 cells after 2 hrs by microplate scintillation and luminescence counting
Displacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation countingDisplacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation counting
Displacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation countingDisplacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation counting
Displacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation countingDisplacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation counting
Displacement of [125I]MCP-1 from human CCR2b expressed in human monocyte cellsDisplacement of [125I]MCP-1 from human CCR2b expressed in human monocyte cells
Displacement of [125I]MCP1 from human CCR2 I40L mutant expressed in CHO cellsDisplacement of [125I]MCP1 from human CCR2 I40L mutant expressed in CHO cells
Displacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assay
Displacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assay
Inhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperatureInhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperature
Displacement of [125I]MCP1 from CCR2 in human PBMC by millipore filter plate assayDisplacement of [125I]MCP1 from CCR2 in human PBMC by millipore filter plate assay
Antagonist activity at CCR2 in human monocytes by chemotaxis assay in presence of 0.5% bovine serum albuminAntagonist activity at CCR2 in human monocytes by chemotaxis assay in presence of 0.5% bovine serum albumin
Antagonist activity at CCR2 in human PBMCs assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMCs assessed as inhibition of MCP1-induced chemotaxis
Displacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assay
Antagonist activity at human CCR2 in PBMCs assessed as inhibition of chemotaxisAntagonist activity at human CCR2 in PBMCs assessed as inhibition of chemotaxis
Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).
Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).
Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).
Displacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assay
Antagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Displacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cellsDisplacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cells
Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).
Displacement of [125I]MCP1 from human CCR2 I40L mutant expressed in CHO cellsDisplacement of [125I]MCP1 from human CCR2 I40L mutant expressed in CHO cells
Displacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assay
Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Antagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis
Antagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Antagonist activity at human CCR2 expressed in THP1 cells assessed as inhibition of MCP-1 induced chemotaxisAntagonist activity at human CCR2 expressed in THP1 cells assessed as inhibition of MCP-1 induced chemotaxis
Antagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxis
Antagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Antagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Antagonist activity at mouse CCR2 assessed as inhibition of MCP1 induced chemotaxis after 30 minsAntagonist activity at mouse CCR2 assessed as inhibition of MCP1 induced chemotaxis after 30 mins
Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).
Binding Assay: THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30° C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 μL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.). Blank values (buffer only) were subtracted from all values and drug treated values were compared to vehicle treated values. 1 μM cold MCP-1 was used for nonspecific binding.Binding Assay: THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30° C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 μL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.). Blank values (buffer only) were subtracted from all values and drug treated values were compared to vehicle treated values. 1 μM cold MCP-1 was used for nonspecific binding.
Displacement of [125I]MCP1 from CCR2 in human PBMC after 30 mins by gamma counterDisplacement of [125I]MCP1 from CCR2 in human PBMC after 30 mins by gamma counter
Displacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assay
Displacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assay
Antagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Antagonist activity at human CCR2b expressed in human Chem1 cells assessed as inhibition of human recombinant CCL2 induced Ca2+ mobilization preincubated for 10 mins followed by CCL2 induction measured every sec for 120 secs by Fluo-4-AM dye-based fluorescence assayAntagonist activity at human CCR2b expressed in human Chem1 cells assessed as inhibition of human recombinant CCL2 induced Ca2+ mobilization preincubated for 10 mins followed by CCL2 induction measured every sec for 120 secs by Fluo-4-AM dye-based fluorescence assay
Displacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation countingDisplacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation counting
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Antagonist activity against human CCR2 receptor assessed as inhibition of MCP1-induced migration of human peripheral blood monocytesAntagonist activity against human CCR2 receptor assessed as inhibition of MCP1-induced migration of human peripheral blood monocytes
Antagonist activity at CCR2 in mouse spleen cells assessed as inhibition of CCL2-induced chemotaxisAntagonist activity at CCR2 in mouse spleen cells assessed as inhibition of CCL2-induced chemotaxis
Displacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation countingDisplacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation counting
Concentration required to inhibit [125I]MCP-1 binding to Chemokine receptor 2BConcentration required to inhibit [125I]MCP-1 binding to Chemokine receptor 2B
Displacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cellsDisplacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cells
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Displacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assayDisplacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assay
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).
Antagonist activity against human CCR2 in human THP1 cells using [I125]MCP-1 by scintillation countingAntagonist activity against human CCR2 in human THP1 cells using [I125]MCP-1 by scintillation counting
Antagonist activity against human CCR2 in human THP1 cells using [I125]MCP-1 by scintillation countingAntagonist activity against human CCR2 in human THP1 cells using [I125]MCP-1 by scintillation counting
Antagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced inhibition of calcium fluxAntagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced inhibition of calcium flux
Antagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Antagonist activity at CCR2 receptor in human PBMC assessed as inhibition of MCP1-mediated leukocyte chemotaxis after 30 mins by microscopyAntagonist activity at CCR2 receptor in human PBMC assessed as inhibition of MCP1-mediated leukocyte chemotaxis after 30 mins by microscopy
Antagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Antagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Displacement of [125I]-MCP1 from CCR2 in human THP1 cells after 2 hrs by microplate scintillation and luminescence countingDisplacement of [125I]-MCP1 from CCR2 in human THP1 cells after 2 hrs by microplate scintillation and luminescence counting
Displacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation countingDisplacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation counting
Displacement of [125I]MCP-1 from human CCR2b expressed in human monocyte cellsDisplacement of [125I]MCP-1 from human CCR2b expressed in human monocyte cells
Displacement of [125I]MCP1 from CCR2 in human PBMC measured after 30 mins by Microscintillation counting analysisDisplacement of [125I]MCP1 from CCR2 in human PBMC measured after 30 mins by Microscintillation counting analysis
Displacement of [125I]MCP1 from CCR2 in human PBMC measured after 30 mins by Microscintillation counting analysisDisplacement of [125I]MCP1 from CCR2 in human PBMC measured after 30 mins by Microscintillation counting analysis
Inhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperatureInhibition of [125I]hMCP1 binding to CCR2 in human PBMC incubated for 30 mins at room temperature
Displacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assay
Displacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assay
Displacement of [125I]MCP1 from human CCR2 I40L mutant expressed in CHO cellsDisplacement of [125I]MCP1 from human CCR2 I40L mutant expressed in CHO cells
Displacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assay
Displacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assay
Displacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assay
Antagonist activity at human CCR2 expressed in HEK293 cells assessed as inhibition of beta-arrestin recruitment pre-incubated for 20 mins before CCL2 stimulation and followed by further incubation for 90 mins by NanoBiT CCR2 assay basedAntagonist activity at human CCR2 expressed in HEK293 cells assessed as inhibition of beta-arrestin recruitment pre-incubated for 20 mins before CCL2 stimulation and followed by further incubation for 90 mins by NanoBiT CCR2 assay based
Antagonist activity at CCR2 in human PBMCs assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMCs assessed as inhibition of MCP1-induced chemotaxis
Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).
Displacement of [125I]MCP-1 from CCR2 in human PBMC by radioligand displacement assayDisplacement of [125I]MCP-1 from CCR2 in human PBMC by radioligand displacement assay
Antagonist activity at CCR2 in human THP1 cells assessed as inhibition of CCL2-induced chemotaxisAntagonist activity at CCR2 in human THP1 cells assessed as inhibition of CCL2-induced chemotaxis
Antagonist activity at CCR2 in human THP1 cells assessed as inhibition of CCL2-induced chemotaxisAntagonist activity at CCR2 in human THP1 cells assessed as inhibition of CCL2-induced chemotaxis
Concentration required to inhibit [125I]MCP-1 (Monocyte Chemoattractant Protein-1) binding to THP-1 cell; not determinedConcentration required to inhibit [125I]MCP-1 (Monocyte Chemoattractant Protein-1) binding to THP-1 cell; not determined
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Displacement of [125I]MCP1 from CCR2 in human PBMC by millipore filter plate assayDisplacement of [125I]MCP1 from CCR2 in human PBMC by millipore filter plate assay
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Antagonist activity against human CCR2 receptor assessed as inhibition of MCP1-induced migration of human peripheral blood monocytesAntagonist activity against human CCR2 receptor assessed as inhibition of MCP1-induced migration of human peripheral blood monocytes
Antagonist activity at mouse CCR2 assessed as inhibition of MCP1-induced chemotaxis after 30 minsAntagonist activity at mouse CCR2 assessed as inhibition of MCP1-induced chemotaxis after 30 mins
Antagonist activity at CCR2 (unknown origin) expressed in CHOK1 cells co-expressing Ga16 assessed as inhibition of CCL2 induced intracellular Ca2+ mobilization preincubated for 10 mins followed by CCL2 addition by fluo-4 AM dye based assayAntagonist activity at CCR2 (unknown origin) expressed in CHOK1 cells co-expressing Ga16 assessed as inhibition of CCL2 induced intracellular Ca2+ mobilization preincubated for 10 mins followed by CCL2 addition by fluo-4 AM dye based assay
Antagonist activity at mouse CCR2 assessed as inhibition of MCP1 induced chemotaxis after 30 minsAntagonist activity at mouse CCR2 assessed as inhibition of MCP1 induced chemotaxis after 30 mins
Antagonist activity against CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis at 37 degCAntagonist activity against CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis at 37 degC
Antagonist activity at CCR2 in human PBMC assessed as MCP1-induced calcium flux by fluorescence-imaging plate reader assayAntagonist activity at CCR2 in human PBMC assessed as MCP1-induced calcium flux by fluorescence-imaging plate reader assay
Antagonist activity at CCR2b in human THP1 cells assessed as inhibition of human MCP1 induced calcium flux by fluo-4 dye based fluorescence assayAntagonist activity at CCR2b in human THP1 cells assessed as inhibition of human MCP1 induced calcium flux by fluo-4 dye based fluorescence assay
Antagonist activity at CCR2b in human THP1 cells assessed as inhibition of human MCP1 induced calcium flux by fluo-4 dye based fluorescence assayAntagonist activity at CCR2b in human THP1 cells assessed as inhibition of human MCP1 induced calcium flux by fluo-4 dye based fluorescence assay
Antagonist activity at human CCR2 expressed in HEK293 cells assessed as inhibition of beta-arrestin recruitment pre-incubated for 20 mins before CCL2 stimulation and followed by further incubation for 90 mins by NanoBiT CCR2 assay basedAntagonist activity at human CCR2 expressed in HEK293 cells assessed as inhibition of beta-arrestin recruitment pre-incubated for 20 mins before CCL2 stimulation and followed by further incubation for 90 mins by NanoBiT CCR2 assay based
Antagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxis
Binding Assay: THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30° C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 μL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.). Blank values (buffer only) were subtracted from all values and drug treated values were compared to vehicle treated values. 1 μM cold MCP-1 was used for nonspecific binding.Binding Assay: THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30° C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 μL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.). Blank values (buffer only) were subtracted from all values and drug treated values were compared to vehicle treated values. 1 μM cold MCP-1 was used for nonspecific binding.
Binding Assay: THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30° C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 μL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.). Blank values (buffer only) were subtracted from all values and drug treated values were compared to vehicle treated values. 1 μM cold MCP-1 was used for nonspecific binding.Binding Assay: THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30° C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 μL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.). Blank values (buffer only) were subtracted from all values and drug treated values were compared to vehicle treated values. 1 μM cold MCP-1 was used for nonspecific binding.
Displacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assayDisplacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assay
Displacement of [125I]-MCP-1 from CCR2 in human THP-1 cells after 2 hrs by microplate scintillation and luminescence counter analysisDisplacement of [125I]-MCP-1 from CCR2 in human THP-1 cells after 2 hrs by microplate scintillation and luminescence counter analysis
Displacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation countingDisplacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation counting
Displacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation countingDisplacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation counting
Displacement of [125I]MCP1 from human CCR2 I40L mutant expressed in CHO cellsDisplacement of [125I]MCP1 from human CCR2 I40L mutant expressed in CHO cells
Antagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxis
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Antagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Antagonist activity at human CCR2 in PBMCs assessed as inhibition of chemotaxisAntagonist activity at human CCR2 in PBMCs assessed as inhibition of chemotaxis
Antagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium levelAntagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium level
Antagonist activity against human CCR2B expressed in Chem-1 cells assessed as inhibition of human CCL2-induced intracellular calcium flux incubated for 10 mins by Fluo-4 AM dye based intracellular calcium flux assayAntagonist activity against human CCR2B expressed in Chem-1 cells assessed as inhibition of human CCL2-induced intracellular calcium flux incubated for 10 mins by Fluo-4 AM dye based intracellular calcium flux assay
Antagonist activity at CCR2 in human THP1 cells assessed as inhibition of CCL2-induced chemotaxisAntagonist activity at CCR2 in human THP1 cells assessed as inhibition of CCL2-induced chemotaxis
Displacement of [125I]MCP-1 from CCR2 in human PBMC by radioligand displacement assayDisplacement of [125I]MCP-1 from CCR2 in human PBMC by radioligand displacement assay
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Antagonist activity at human CCR2 receptor expressed in human THP-1 cells assessed as inhibition of MCP1-induced Ca2+ flux by FLIPR analysisAntagonist activity at human CCR2 receptor expressed in human THP-1 cells assessed as inhibition of MCP1-induced Ca2+ flux by FLIPR analysis
Antagonist activity at CCR2 (unknown origin) expressed in CHOK1 cells co-expressing Ga16 assessed as inhibition of CCL2 induced intracellular Ca2+ mobilization preincubated for 10 mins followed by CCL2 addition by fluo-4 AM dye based assayAntagonist activity at CCR2 (unknown origin) expressed in CHOK1 cells co-expressing Ga16 assessed as inhibition of CCL2 induced intracellular Ca2+ mobilization preincubated for 10 mins followed by CCL2 addition by fluo-4 AM dye based assay
Antagonist activity at human CCR2 receptor expressed in human THP-1 cells assessed as inhibition of MCP1-induced Ca2+ flux by FLIPR analysisAntagonist activity at human CCR2 receptor expressed in human THP-1 cells assessed as inhibition of MCP1-induced Ca2+ flux by FLIPR analysis
Antagonist activity against human CCR2B expressed in Chem-1 cells assessed as inhibition of human CCL2-induced intracellular calcium flux incubated for 10 mins by Fluo-4 AM dye based intracellular calcium flux assayAntagonist activity against human CCR2B expressed in Chem-1 cells assessed as inhibition of human CCL2-induced intracellular calcium flux incubated for 10 mins by Fluo-4 AM dye based intracellular calcium flux assay
Displacement of 125I-hMCP1 from CCR2 expressed in human peripheral blood monocytes after 60 minsDisplacement of 125I-hMCP1 from CCR2 expressed in human peripheral blood monocytes after 60 mins
Displacement of [125I]MCP-1 from CCR2 in human PBMC by radioligand displacement assayDisplacement of [125I]MCP-1 from CCR2 in human PBMC by radioligand displacement assay
Antagonist activity against CCR2 in human THP-1 cells assessed as inhibition of MCP-1 induced chemotaxis after 3 hrsAntagonist activity against CCR2 in human THP-1 cells assessed as inhibition of MCP-1 induced chemotaxis after 3 hrs
Antagonist activity against human CCR2 in human THP1 cells using [I125]MCP-1 by scintillation countingAntagonist activity against human CCR2 in human THP1 cells using [I125]MCP-1 by scintillation counting
Antagonist activity against human CCR2B expressed in Chem-1 cells assessed as inhibition of human CCL2-induced intracellular calcium flux incubated for 10 mins by Fluo-4 AM dye based intracellular calcium flux assayAntagonist activity against human CCR2B expressed in Chem-1 cells assessed as inhibition of human CCL2-induced intracellular calcium flux incubated for 10 mins by Fluo-4 AM dye based intracellular calcium flux assay
Antagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMC assessed as inhibition of MCP1-induced chemotaxis
Antagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Displacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assayDisplacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assay
Displacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assayDisplacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assay
Displacement of MCP-Alexa 488 from CCR2 in human whole blood after 5 mins by flow cytometryDisplacement of MCP-Alexa 488 from CCR2 in human whole blood after 5 mins by flow cytometry
Displacement of MCP-Alexa 488 from CCR2 in human whole blood after 5 mins by flow cytometryDisplacement of MCP-Alexa 488 from CCR2 in human whole blood after 5 mins by flow cytometry
Displacement of [125I]MCP1 from human CCR2 preincubated 1 hrs by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated 1 hrs by human whole cell binding assay
Displacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assay
Antagonist activity at CCR2 in human PBMCs assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMCs assessed as inhibition of MCP1-induced chemotaxis
Antagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxisAntagonist activity at CCR2 in human PBMC assessed as MCP1-induced chemotaxis
Displacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated 30 mins by human whole cell binding assay
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Displacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assay
Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).Radioligand Binding Assay: Radioligand binding assays were performed in CHO cells expressing human CCR2B and Galpha±16 coupling protein. All compounds were dissolved in DMSO and assays run at a final DMSO concentration of 0.5% (v/v). [125I]-labeled human MCP-1 was purchased from PerkinElmer. Unlabelled human MCP-1 was purchased from PeproTech.Compounds were serially diluted in DMSO before diluting into assay buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA) with cryo preserved CHO cells expressing human CCR2B and the Galpha±16 coupling protein (30x103/well) and [125I]-MCP-1 (50 pM). The reaction was incubated at room temperature for 90 minutes before transferring to GF/C filter membrane (PerkinElmer) pre-treated with 0.3% polyethyleneimine for 2 hours at 4 C. The filter membrane was washed six times with ice cold wash buffer (25 mM HEPES, pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 mM NaCl, 0.01% (m/v) azide), dried, and sealed in a RLB sample bag (Agilent Technologies).
Antagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Antagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at CCR2 in human THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Antagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrsAntagonist activity at human CCR2 receptor in THP1 cells assessed as inhibition of MCP1-induced chemotaxis after 3 hrs
Antagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium levelAntagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium level
Binding Assay: THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30° C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 μL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.). Blank values (buffer only) were subtracted from all values and drug treated values were compared to vehicle treated values. 1 μM cold MCP-1 was used for nonspecific binding.Binding Assay: THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30° C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 μL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.). Blank values (buffer only) were subtracted from all values and drug treated values were compared to vehicle treated values. 1 μM cold MCP-1 was used for nonspecific binding.
Displacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assayDisplacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assay
Displacement of [125I]-MCP-1 from CCR2 in human THP-1 cells after 2 hrs by microplate scintillation and luminescence counter analysisDisplacement of [125I]-MCP-1 from CCR2 in human THP-1 cells after 2 hrs by microplate scintillation and luminescence counter analysis
Displacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation countingDisplacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation counting
Concentration required to inhibit [125I]MCP-1 binding to Chemokine receptor 2BConcentration required to inhibit [125I]MCP-1 binding to Chemokine receptor 2B
Concentration required to inhibit [125I]MCP-1 binding to Chemokine receptor 2BConcentration required to inhibit [125I]MCP-1 binding to Chemokine receptor 2B
Displacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assayDisplacement of 125I-MCP1 from human CCR2 receptor expressed in human THP-1 cell membrane by SPA assay
Displacement of [125I]MCP1 from CCR2 in human PBMC by millipore filter plate assayDisplacement of [125I]MCP1 from CCR2 in human PBMC by millipore filter plate assay
Antagonist activity against human CCR2B expressed in Chem-1 cells assessed as inhibition of human CCL2-induced intracellular calcium flux incubated for 10 mins by Fluo-4 AM dye based intracellular calcium flux assayAntagonist activity against human CCR2B expressed in Chem-1 cells assessed as inhibition of human CCL2-induced intracellular calcium flux incubated for 10 mins by Fluo-4 AM dye based intracellular calcium flux assay
Displacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cellsDisplacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cells
Antagonist activity at CCR2 (unknown origin) expressed in CHOK1 cells co-expressing Ga16 assessed as inhibition of CCL2 induced intracellular Ca2+ mobilization preincubated for 10 mins followed by CCL2 addition by fluo-4 AM dye based assayAntagonist activity at CCR2 (unknown origin) expressed in CHOK1 cells co-expressing Ga16 assessed as inhibition of CCL2 induced intracellular Ca2+ mobilization preincubated for 10 mins followed by CCL2 addition by fluo-4 AM dye based assay
Antagonist activity at human CCR2 receptor expressed in human THP-1 cells assessed as inhibition of MCP1-induced Ca2+ flux by FLIPR analysisAntagonist activity at human CCR2 receptor expressed in human THP-1 cells assessed as inhibition of MCP1-induced Ca2+ flux by FLIPR analysis
Displacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cellsDisplacement of [125I]MCP1 from human CCR2B receptor expressed in HEK293-EBNA cells
Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).Binding Assay: MCP-1 Receptor Binding Assay in THP-1 Cells Human monocytic cell line THP-1 cells were obtained from American Type Culture Collection (Manassas, Va., USA). The THP-1 cells were grown in RPMI-1640 (RPMI: Roswell Park Memorial Institute Medium-cell culture growth media) supplemented with 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37 C. The cell density was maintained between 0.5x106 cells/mL.THP-1 (cells were incubated with 0.5 nM 125I labeled MCP-1 (Perkin-Elmer Life Sciences, Inc. Boston, Mass.) in the presence of varying concentrations of either unlabeled MCP-1 (R & D Systems, Minneapolis, Minn.) or test compound for 2 hours at 30 C. in a 96 well plate. Cells were then harvested onto a filter plate, dried, and 20 uL of Microscint 20 was added to each well. Plates were counted in a TopCount NXT, Microplate Scintillation & Luminescence Counter (Perkin-Elmer Life Sciences, Inc. Boston, Mass.).
Antagonist activity against human CCR2 receptor assessed as inhibition of MCP1-induced migration of human peripheral blood monocytesAntagonist activity against human CCR2 receptor assessed as inhibition of MCP1-induced migration of human peripheral blood monocytes
Antagonist activity at CCR2 in human PBMC assessed as MCP1-induced calcium flux by fluorescence-imaging plate reader assayAntagonist activity at CCR2 in human PBMC assessed as MCP1-induced calcium flux by fluorescence-imaging plate reader assay
Antagonist activity at CCR2 receptor in human PBMC assessed as inhibition of MCP1-mediated leukocyte chemotaxis after 30 mins by microscopyAntagonist activity at CCR2 receptor in human PBMC assessed as inhibition of MCP1-mediated leukocyte chemotaxis after 30 mins by microscopy
Antagonist activity at CCR2 receptor in human THP1 cells assessed as inhibition of MCP-1 induced chemotaxis after 3 hrsAntagonist activity at CCR2 receptor in human THP1 cells assessed as inhibition of MCP-1 induced chemotaxis after 3 hrs
Antagonist activity at human CCR2 in PBMCs assessed as inhibition of MCP1-induced calcium fluxAntagonist activity at human CCR2 in PBMCs assessed as inhibition of MCP1-induced calcium flux
Antagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxisAntagonist activity at human CCR2 in THP1 cells assessed as inhibition of MCP1-induced chemotaxis
Antagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium levelAntagonist activity at human CCR2B receptor expressed in HEK293 cells assessed as inhibition of MCP1-induced increase in intracellular calcium level
Displacement of [125I]-MCP-1 from CCR2 in human THP-1 cells after 2 hrs by microplate scintillation and luminescence counter analysisDisplacement of [125I]-MCP-1 from CCR2 in human THP-1 cells after 2 hrs by microplate scintillation and luminescence counter analysis
Displacement of [125I]-MCP-1 from CCR2 in human THP-1 cells after 2 hrs by microplate scintillation and luminescence counter analysisDisplacement of [125I]-MCP-1 from CCR2 in human THP-1 cells after 2 hrs by microplate scintillation and luminescence counter analysis
Displacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation countingDisplacement of [125I]-MCP1 from human CCR2 receptor in THP1 cells after 2 hrs by scintillation counting
Displacement of [125I]MCP1 from human CCR2 in PBMC after 30 mins by gamma countingDisplacement of [125I]MCP1 from human CCR2 in PBMC after 30 mins by gamma counting
Antagonist activity at human CCR2 assessed as inhibition of MCP1-induced chemotaxis by cell based assayAntagonist activity at human CCR2 assessed as inhibition of MCP1-induced chemotaxis by cell based assay
Displacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assayDisplacement of [125I]MCP1 from human CCR2 preincubated for 30 mins by human whole cell binding assay
Antagonist activity against human CCR2B expressed in Chem-1 cells assessed as inhibition of human CCL2-induced intracellular calcium flux incubated for 10 mins by Fluo-4 AM dye based intracellular calcium flux assayAntagonist activity against human CCR2B expressed in Chem-1 cells assessed as inhibition of human CCL2-induced intracellular calcium flux incubated for 10 mins by Fluo-4 AM dye based intracellular calcium flux assay