Agonist activity at rat mu-opioid receptor expressed in HEK293 cells co-expressing Rluc2-EPAC-GFP10 biosenser assessed as inhibition of forskolin-induced cAMP level after 10 mins by fluorescence based assayAgonist activity at rat mu-opioid receptor expressed in HEK293 cells co-expressing Rluc2-EPAC-GFP10 biosenser assessed as inhibition of forskolin-induced cAMP level after 10 mins by fluorescence based assay
Agonist activity at rat mu-opioid receptor expressed in HEK293 cells co-expressing Rluc2-EPAC-GFP10 biosenser assessed as inhibition of forskolin-induced cAMP level after 10 mins by fluorescence based assayAgonist activity at rat mu-opioid receptor expressed in HEK293 cells co-expressing Rluc2-EPAC-GFP10 biosenser assessed as inhibition of forskolin-induced cAMP level after 10 mins by fluorescence based assay
Agonist activity at human MOR expressed in CHO-K1 cells assessed as cAMP accumulation incubated for 30 mins and measured after 1 hr by Eu-cAMP tracer based TR-FRET assayAgonist activity at human MOR expressed in CHO-K1 cells assessed as cAMP accumulation incubated for 30 mins and measured after 1 hr by Eu-cAMP tracer based TR-FRET assay
Agonist activity at human MOR expressed in CHOK1 cells assessed as inhibition of forskolin-stimulated cAMP accumulation incubated for 30 mins by luminescence assayAgonist activity at human MOR expressed in CHOK1 cells assessed as inhibition of forskolin-stimulated cAMP accumulation incubated for 30 mins by luminescence assay
Agonist activity at human MOR expressed in CHO-K1 cells assessed as cAMP accumulation incubated for 30 mins and measured after 1 hr by Eu-cAMP tracer based TR-FRET assayAgonist activity at human MOR expressed in CHO-K1 cells assessed as cAMP accumulation incubated for 30 mins and measured after 1 hr by Eu-cAMP tracer based TR-FRET assay
Agonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counterAgonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counter
Agonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counterAgonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counter
Agonist activity at FLAG-tagged mu-type opioid receptor (unknown origin) expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation after 30 mins by liquid scintillation counting analysisAgonist activity at FLAG-tagged mu-type opioid receptor (unknown origin) expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation after 30 mins by liquid scintillation counting analysis
Activity at mu opioid receptor assessed as increase in calcium level in CHO cells by aequorin luminescence based calcium assayActivity at mu opioid receptor assessed as increase in calcium level in CHO cells by aequorin luminescence based calcium assay
Activity at mu opioid receptor assessed as increase in calcium level in CHO cells by aequorin luminescence based calcium assayActivity at mu opioid receptor assessed as increase in calcium level in CHO cells by aequorin luminescence based calcium assay
Agonist activity at rat mu-opioid receptor expressed in HEK293 cells co-expressing Rluc2-EPAC-GFP10 biosenser assessed as inhibition of forskolin-induced cAMP level after 10 mins by fluorescence based assayAgonist activity at rat mu-opioid receptor expressed in HEK293 cells co-expressing Rluc2-EPAC-GFP10 biosenser assessed as inhibition of forskolin-induced cAMP level after 10 mins by fluorescence based assay
Antagonist activity at mu opioid receptor expressed in CHO cells assessed as release of intracellular calcium ions by aequorin luminescence-based calcium assayAntagonist activity at mu opioid receptor expressed in CHO cells assessed as release of intracellular calcium ions by aequorin luminescence-based calcium assay
Antagonist activity at mu opioid receptor expressed in CHO cells assessed as release of intracellular calcium ions by aequorin luminescence-based calcium assayAntagonist activity at mu opioid receptor expressed in CHO cells assessed as release of intracellular calcium ions by aequorin luminescence-based calcium assay
Agonist activity at FLAG-tagged mu-type opioid receptor (unknown origin) expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation after 30 mins by liquid scintillation counting analysisAgonist activity at FLAG-tagged mu-type opioid receptor (unknown origin) expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation after 30 mins by liquid scintillation counting analysis
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at mu opioid receptor (unknown origin) transfected in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP productionAgonist activity at mu opioid receptor (unknown origin) transfected in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP production
Agonist activity at mu opioid receptor (unknown origin) transfected in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP productionAgonist activity at mu opioid receptor (unknown origin) transfected in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP production
Agonist activity at human MOR expressed in HEK293 cell membrane assessed as inhibition of forskolin-induced cAMP accumulation by by liquid scintillation countingAgonist activity at human MOR expressed in HEK293 cell membrane assessed as inhibition of forskolin-induced cAMP accumulation by by liquid scintillation counting
Agonist activity at human MOR expressed in HEK293 cell membrane assessed as inhibition of forskolin-induced cAMP accumulation by by liquid scintillation countingAgonist activity at human MOR expressed in HEK293 cell membrane assessed as inhibition of forskolin-induced cAMP accumulation by by liquid scintillation counting
Activity at monocloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayActivity at monocloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at FLAG-tagged mu-type opioid receptor (unknown origin) expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation after 30 mins by liquid scintillation counting analysisAgonist activity at FLAG-tagged mu-type opioid receptor (unknown origin) expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation after 30 mins by liquid scintillation counting analysis
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assayAgonist activity at human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assay
Agonist activity at human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assayAgonist activity at human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assay
Agonist activity at human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assayAgonist activity at human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assay
Agonist activity at human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assayAgonist activity at human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assay
Agonist activity at human MOR stably expressed in CHO cell membranes assessed as stimulation of [35S]-GTPgammaS binding incubated for 1 hr by liquid scintillation counting assayAgonist activity at human MOR stably expressed in CHO cell membranes assessed as stimulation of [35S]-GTPgammaS binding incubated for 1 hr by liquid scintillation counting assay
Agonist activity at rat mu-opioid receptor expressed in HEK293 cells co-expressing Rluc2-EPAC-GFP10 biosenser assessed as inhibition of forskolin-induced cAMP level after 10 mins by fluorescence based assayAgonist activity at rat mu-opioid receptor expressed in HEK293 cells co-expressing Rluc2-EPAC-GFP10 biosenser assessed as inhibition of forskolin-induced cAMP level after 10 mins by fluorescence based assay
Activity at human cloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayActivity at human cloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Activity at human cloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayActivity at human cloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu-opioid receptor expressed in HEK293 cells co-expressing Rluc2-EPAC-GFP10 biosenser assessed as inhibition of forskolin-induced cAMP level after 10 mins by fluorescence based assayAgonist activity at rat mu-opioid receptor expressed in HEK293 cells co-expressing Rluc2-EPAC-GFP10 biosenser assessed as inhibition of forskolin-induced cAMP level after 10 mins by fluorescence based assay
Agonist activity at rat mu-opioid receptor expressed in HEK293 cells co-expressing Rluc2-EPAC-GFP10 biosenser assessed as inhibition of forskolin-induced cAMP level after 10 mins by fluorescence based assayAgonist activity at rat mu-opioid receptor expressed in HEK293 cells co-expressing Rluc2-EPAC-GFP10 biosenser assessed as inhibition of forskolin-induced cAMP level after 10 mins by fluorescence based assay
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at recombinant human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assayAgonist activity at recombinant human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assay
Agonist activity at human mu opioid receptor expressed in HEK293 cells by CellKey methodAgonist activity at human mu opioid receptor expressed in HEK293 cells by CellKey method
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Partial agonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding after 60 mins by scintillation countingPartial agonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding after 60 mins by scintillation counting
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
Agonist activity at mouse mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding after 1.5 hrsAgonist activity at mouse mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding after 1.5 hrs
Agonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counterAgonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counter
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Stimulation of mouse MOR expressed in CHO cells after 1.5 hrs by [35S]GTPgammaS binding assayStimulation of mouse MOR expressed in CHO cells after 1.5 hrs by [35S]GTPgammaS binding assay
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membrane assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in rat C6 cell membrane assessed as stimulation of [35S]GTPgammaS binding
Compound was tested for its ability to stimulate [35S]GTP-gamma-S, binding to membranes from C6 glioma cells stably expressing rat mu opioid receptor.Compound was tested for its ability to stimulate [35S]GTP-gamma-S, binding to membranes from C6 glioma cells stably expressing rat mu opioid receptor.
Agonist activity at human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assayAgonist activity at human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assay
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at human MOR expressed in CHO-K1 cells assessed as cAMP accumulation incubated for 30 mins and measured after 1 hr by Eu-cAMP tracer based TR-FRET assayAgonist activity at human MOR expressed in CHO-K1 cells assessed as cAMP accumulation incubated for 30 mins and measured after 1 hr by Eu-cAMP tracer based TR-FRET assay
Agonist activity at human MOR expressed in CHOK1 cells assessed as stimulation of cAMP accumulation incubated for 45 mins by HTRF assayAgonist activity at human MOR expressed in CHOK1 cells assessed as stimulation of cAMP accumulation incubated for 45 mins by HTRF assay
Agonist activity at human recombinant MOP receptor expressed in CHO-K1 cells assessed as stimulation of [35S]GTPgammaS binding by scintillation proximity assayAgonist activity at human recombinant MOP receptor expressed in CHO-K1 cells assessed as stimulation of [35S]GTPgammaS binding by scintillation proximity assay
Agonist activity at human recombinant MOP receptor expressed in CHO-K1 cells assessed as stimulation of [35S]GTPgammaS binding by scintillation proximity assayAgonist activity at human recombinant MOP receptor expressed in CHO-K1 cells assessed as stimulation of [35S]GTPgammaS binding by scintillation proximity assay
Agonist activity at rat mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assayAgonist activity at rat mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assay
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding
Partial agonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding after 60 mins by scintillation countingPartial agonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding after 60 mins by scintillation counting
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at human MOR expressed in CHOK1 cells assessed as inhibition of forskolin-stimulated cAMP accumulation incubated for 30 mins by luminescence assayAgonist activity at human MOR expressed in CHOK1 cells assessed as inhibition of forskolin-stimulated cAMP accumulation incubated for 30 mins by luminescence assay
Agonist activity at rat MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr by liquid scintillation counting analysisAgonist activity at rat MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr by liquid scintillation counting analysis
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysisAgonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysis
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu-opioid receptor expressed in HEK293 cells co-expressing Rluc2-EPAC-GFP10 biosenser assessed as inhibition of forskolin-induced cAMP level after 10 mins by fluorescence based assayAgonist activity at rat mu-opioid receptor expressed in HEK293 cells co-expressing Rluc2-EPAC-GFP10 biosenser assessed as inhibition of forskolin-induced cAMP level after 10 mins by fluorescence based assay
Agonist activity at rat mu-opioid receptor expressed in HEK293 cells co-expressing Rluc2-EPAC-GFP10 biosenser assessed as inhibition of forskolin-induced cAMP level after 10 mins by fluorescence based assayAgonist activity at rat mu-opioid receptor expressed in HEK293 cells co-expressing Rluc2-EPAC-GFP10 biosenser assessed as inhibition of forskolin-induced cAMP level after 10 mins by fluorescence based assay
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Activity at monocloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayActivity at monocloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assayAgonist activity at human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assay
Agonist activity at human MOR expressed in CHO-K1 cells assessed as cAMP accumulation incubated for 30 mins and measured after 1 hr by Eu-cAMP tracer based TR-FRET assayAgonist activity at human MOR expressed in CHO-K1 cells assessed as cAMP accumulation incubated for 30 mins and measured after 1 hr by Eu-cAMP tracer based TR-FRET assay
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at human MOR expressed in CHOK1 cells assessed as inhibition of forskolin-stimulated cAMP accumulation incubated for 30 mins by luminescence assayAgonist activity at human MOR expressed in CHOK1 cells assessed as inhibition of forskolin-stimulated cAMP accumulation incubated for 30 mins by luminescence assay
Agonist activity at human MOR expressed in CHOK1 cells assessed as inhibition of forskolin-stimulated cAMP accumulation incubated for 30 mins by luminescence assayAgonist activity at human MOR expressed in CHOK1 cells assessed as inhibition of forskolin-stimulated cAMP accumulation incubated for 30 mins by luminescence assay
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding
Agonist activity at human mu-opioid receptor expressed in HEK293 cell membranes after 1 hr by [35S]-GTPgammaS binding assayAgonist activity at human mu-opioid receptor expressed in HEK293 cell membranes after 1 hr by [35S]-GTPgammaS binding assay
Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at recombinant human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assayAgonist activity at recombinant human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assay
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Activity at monocloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayActivity at monocloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
Agonist activity at mu opioid receptor (unknown origin) expressed CHO cells assessed as stimulation of [35S]-GTP[gammaS] bindingAgonist activity at mu opioid receptor (unknown origin) expressed CHO cells assessed as stimulation of [35S]-GTP[gammaS] binding
Agonist activity at rat MOR transfected in rat C6 cell membranes after 1 hr by [35S]GTPgammaS assayAgonist activity at rat MOR transfected in rat C6 cell membranes after 1 hr by [35S]GTPgammaS assay
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.
Agonist activity at EA-fragment beta-arrestin-tagged human MOR expressed in human U2OS cell membranes assessed as stimulation of [35S]-GTPgammaS binding after 60 mins by beta-galactosidase based scintillation counting methodAgonist activity at EA-fragment beta-arrestin-tagged human MOR expressed in human U2OS cell membranes assessed as stimulation of [35S]-GTPgammaS binding after 60 mins by beta-galactosidase based scintillation counting method
Agonist activity at human mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayAgonist activity at human mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human mu opioid receptor expressed in CHO membrane assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at human mu opioid receptor expressed in CHO membrane assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at human mu opioid receptor expressed in CHO membrane assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at human mu opioid receptor expressed in CHO membrane assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as maximal stimulation of [35S]GTP-gamma-S bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as maximal stimulation of [35S]GTP-gamma-S binding
μ-Opioid Receptor Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared in-house from a cell line expressing recombinant μ opioid receptor in a HEK293 background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25 °C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50 °C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.μ-Opioid Receptor Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared in-house from a cell line expressing recombinant μ opioid receptor in a HEK293 background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25 °C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50 °C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.
μ-Opioid Receptor Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared in-house from a cell line expressing recombinant μ opioid receptor in a HEK293 background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25 °C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50 °C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.μ-Opioid Receptor Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared in-house from a cell line expressing recombinant μ opioid receptor in a HEK293 background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25 °C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50 °C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.
Activity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cellsActivity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cells
Activity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cellsActivity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cells
Agonist activity at human MOR expressed in CHOK1 cells assessed as inhibition of forskolin-stimulated cAMP accumulation incubated for 30 mins by luminescence assayAgonist activity at human MOR expressed in CHOK1 cells assessed as inhibition of forskolin-stimulated cAMP accumulation incubated for 30 mins by luminescence assay
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysisAgonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysis
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Partial agonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding after 60 mins by scintillation countingPartial agonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding after 60 mins by scintillation counting
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
Agonist activity at human MOR expressed in CHO-K1 cells assessed as cAMP accumulation incubated for 30 mins and measured after 1 hr by Eu-cAMP tracer based TR-FRET assayAgonist activity at human MOR expressed in CHO-K1 cells assessed as cAMP accumulation incubated for 30 mins and measured after 1 hr by Eu-cAMP tracer based TR-FRET assay
Agonist activity at human mu opioid receptor transfected in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation counting analysisAgonist activity at human mu opioid receptor transfected in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation counting analysis
Agonist activity at human mu opioid receptor transfected in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation counting analysisAgonist activity at human mu opioid receptor transfected in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation counting analysis
Agonist activity at human mu opioid receptor transfected in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation counting analysisAgonist activity at human mu opioid receptor transfected in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation counting analysis
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr by liquid scintillation counting analysisAgonist activity at rat MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr by liquid scintillation counting analysis
Agonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysisAgonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysis
Antagonist activity at mouse MOR expressed in CHO cell membranes assessed as inhibition of DAMGO-stimulated [35S]GTPgammaS binding after 90 minsAntagonist activity at mouse MOR expressed in CHO cell membranes assessed as inhibition of DAMGO-stimulated [35S]GTPgammaS binding after 90 mins
Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.
Agonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysisAgonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysis
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at rat mu opioid receptor expressed in mouse HN9.10 cells assessed as [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in mouse HN9.10 cells assessed as [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor expressed in mouse HN9.10 cells assessed as [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in mouse HN9.10 cells assessed as [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assayAgonist activity at rat mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assay
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at human mu opiod receptor expressed in CHO-K1 cells assessed as increase in forskolin induced cAMP production measured after 30 mins by HitHunter luminescence based assayAgonist activity at human mu opiod receptor expressed in CHO-K1 cells assessed as increase in forskolin induced cAMP production measured after 30 mins by HitHunter luminescence based assay
Agonist activity at human mu opiod receptor expressed in CHO-K1 cells assessed as increase in forskolin induced cAMP production measured after 30 mins by HitHunter luminescence based assayAgonist activity at human mu opiod receptor expressed in CHO-K1 cells assessed as increase in forskolin induced cAMP production measured after 30 mins by HitHunter luminescence based assay
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membrane assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in rat C6 cell membrane assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
EC50 for binding of [35S]- GTPdeltaS in cloned human Opioid receptor mu 1 (DAMGO) transfected onto CHO cellsEC50 for binding of [35S]- GTPdeltaS in cloned human Opioid receptor mu 1 (DAMGO) transfected onto CHO cells
Partial agonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding after 60 mins by scintillation countingPartial agonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding after 60 mins by scintillation counting
Agonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysisAgonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysis
Agonist activity at human recombinant MOR expressed in CHO cells co-expressing C-terminally modified Galphaqi5 assessed as induction of calcium mobilization by Fluo-4AM dye-based fluorescence assayAgonist activity at human recombinant MOR expressed in CHO cells co-expressing C-terminally modified Galphaqi5 assessed as induction of calcium mobilization by Fluo-4AM dye-based fluorescence assay
Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.
Inhibition of [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in CHO cellsInhibition of [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in CHO cells
Inhibition of [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in CHO cellsInhibition of [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in CHO cells
EC50 for binding of [35S]- GTPdeltaS in cloned human oOpioid receptor mu 1 (DAMGO) transfected onto CHO cellsEC50 for binding of [35S]- GTPdeltaS in cloned human oOpioid receptor mu 1 (DAMGO) transfected onto CHO cells
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
Activity at monocloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayActivity at monocloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 minsAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 minsAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membrane assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in rat C6 cell membrane assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at mouse mu opioid receptor-1 expressed in CHO cell membranes assessed as [35S]GTPgammaS binding incubated for 60 mins by scintillation spectroscopic analysisAgonist activity at mouse mu opioid receptor-1 expressed in CHO cell membranes assessed as [35S]GTPgammaS binding incubated for 60 mins by scintillation spectroscopic analysis
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor by [35S]GTP-gamma-S binding assayAgonist activity at rat mu opioid receptor by [35S]GTP-gamma-S binding assay
Agonist activity at human mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayAgonist activity at human mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counterAgonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counter
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor expressed in mouse HN9.10 cells assessed as [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in mouse HN9.10 cells assessed as [35S]GTPgammaS binding
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting methodAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting method
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting methodAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting method
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at rat MOR transfected in rat C6 cell membranes after 1 hr by [35S]GTPgammaS assayAgonist activity at rat MOR transfected in rat C6 cell membranes after 1 hr by [35S]GTPgammaS assay
Activity at human mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assayActivity at human mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assay
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding by scintillation countingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding by scintillation counting
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as maximal stimulation of [35S]GTP-gamma-S bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as maximal stimulation of [35S]GTP-gamma-S binding
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [33S]GTPgammaS binding after 60 mins by scintillation countingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [33S]GTPgammaS binding after 60 mins by scintillation counting
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding after 60 minsAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding after 60 mins
Agonist activity at rat MOR expressed in HN9.10 cell membranes by [35S]GTPgammaS binding assayAgonist activity at rat MOR expressed in HN9.10 cell membranes by [35S]GTPgammaS binding assay
Agonistic activity against Opioid receptor mu 1 in Chinese hamster ovary membranesAgonistic activity against Opioid receptor mu 1 in Chinese hamster ovary membranes
GTPgammaS binding in cloned human Opioid receptor mu 1 transfected into hamster ovary cellsGTPgammaS binding in cloned human Opioid receptor mu 1 transfected into hamster ovary cells
Inhibitory activity in stimulating [35S]-GTP-gamma S binding mediated by the Opioid receptor mu 1 in chinese Hamster Ovary (CHO) cell membranes was determinedInhibitory activity in stimulating [35S]-GTP-gamma S binding mediated by the Opioid receptor mu 1 in chinese Hamster Ovary (CHO) cell membranes was determined
Agonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS bindingAgonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding
Agonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS bindingAgonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
Agonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS bindingAgonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS binding
Agonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS bindingAgonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS binding
Agonist activity at rat mu opioid receptor expressed in mouse HN9.10 cells assessed as [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in mouse HN9.10 cells assessed as [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor expressed in mouse HN9.10 cells assessed as [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in mouse HN9.10 cells assessed as [35S]GTPgammaS binding
Agonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counterAgonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counter
Agonist activity at rat MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr by liquid scintillation counting analysisAgonist activity at rat MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr by liquid scintillation counting analysis
Agonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysisAgonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysis
[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Partial agonist activity at human mu opioid receptor expressed in CHO cell membrane assessed as stimulation of [35S]GTPgammaS binding after 60 mins by liquid scintillation countingPartial agonist activity at human mu opioid receptor expressed in CHO cell membrane assessed as stimulation of [35S]GTPgammaS binding after 60 mins by liquid scintillation counting
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
μ-Opioid Receptor Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared in-house from a cell line expressing recombinant μ opioid receptor in a HEK293 background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25 °C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50 °C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.μ-Opioid Receptor Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared in-house from a cell line expressing recombinant μ opioid receptor in a HEK293 background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25 °C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50 °C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu-opioid receptor expressed in CHO cells after 90 mins by [35S]GTP-gamma-S assayAgonist activity at rat mu-opioid receptor expressed in CHO cells after 90 mins by [35S]GTP-gamma-S assay
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu-opioid receptor expressed in CHO cells after 90 mins by [35S]GTP-gamma-S assayAgonist activity at rat mu-opioid receptor expressed in CHO cells after 90 mins by [35S]GTP-gamma-S assay
Agonist activity at rat MOR transfected in rat C6 cell membranes after 1 hr by [35S]GTPgammaS assayAgonist activity at rat MOR transfected in rat C6 cell membranes after 1 hr by [35S]GTPgammaS assay
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 90 mins by liquid scintillation counting analysisAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 90 mins by liquid scintillation counting analysis
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 90 mins by liquid scintillation counting analysisAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 90 mins by liquid scintillation counting analysis
Agonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysisAgonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysis
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at human MOR expressed in CHOK1 cells assessed as inhibition of forskolin-stimulated cAMP accumulation incubated for 30 mins by luminescence assayAgonist activity at human MOR expressed in CHOK1 cells assessed as inhibition of forskolin-stimulated cAMP accumulation incubated for 30 mins by luminescence assay
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at rat mu opioid receptor expressed in mouse HN9.10 cells assessed as [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in mouse HN9.10 cells assessed as [35S]GTPgammaS binding
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.
Activation of human mu opioid receptor expressed in HEK293a cells by [35S]GTP-gamma-S binding assayActivation of human mu opioid receptor expressed in HEK293a cells by [35S]GTP-gamma-S binding assay
Activation of human mu opioid receptor expressed in HEK293a cells by [35S]GTP-gamma-S binding assayActivation of human mu opioid receptor expressed in HEK293a cells by [35S]GTP-gamma-S binding assay
Activation of human mu opioid receptor expressed in HEK293a cells coexpressing YFP-labelled alphai1 and CFP-labelled beta-1-gamma-2 Gi subunits assessed as decrease in fluorescence resonance energy transfer signal at 10 uMActivation of human mu opioid receptor expressed in HEK293a cells coexpressing YFP-labelled alphai1 and CFP-labelled beta-1-gamma-2 Gi subunits assessed as decrease in fluorescence resonance energy transfer signal at 10 uM
Activation of human mu opioid receptor expressed in HEK293a cells coexpressing YFP-labelled alphai1 and CFP-labelled beta-1-gamma-2 Gi subunits assessed as decrease in fluorescence resonance energy transfer signal at 10 uMActivation of human mu opioid receptor expressed in HEK293a cells coexpressing YFP-labelled alphai1 and CFP-labelled beta-1-gamma-2 Gi subunits assessed as decrease in fluorescence resonance energy transfer signal at 10 uM
Activity at human cloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayActivity at human cloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human MOR expressed in CHOK1 cells assessed as stimulation of cAMP accumulation incubated for 45 mins by HTRF assayAgonist activity at human MOR expressed in CHOK1 cells assessed as stimulation of cAMP accumulation incubated for 45 mins by HTRF assay
Agonist activity at human MOR expressed in CHOK1 cells assessed as stimulation of cAMP accumulation incubated for 45 mins by HTRF assayAgonist activity at human MOR expressed in CHOK1 cells assessed as stimulation of cAMP accumulation incubated for 45 mins by HTRF assay
Agonist activity at mu opioid receptor expressed in CHO-hg cells by [35S]-GTPgammaS binding assayAgonist activity at mu opioid receptor expressed in CHO-hg cells by [35S]-GTPgammaS binding assay
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membrane assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in rat C6 cell membrane assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.
Activity at monocloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayActivity at monocloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at mouse mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding after 1.5 hrsAgonist activity at mouse mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding after 1.5 hrs
Stimulation of mouse MOR expressed in CHO cells after 1.5 hrs by [35S]GTPgammaS binding assayStimulation of mouse MOR expressed in CHO cells after 1.5 hrs by [35S]GTPgammaS binding assay
Agonist activity at human mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayAgonist activity at human mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [33S]GTPgammaS binding after 60 mins by scintillation countingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [33S]GTPgammaS binding after 60 mins by scintillation counting
Agonist activity at rat MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr by liquid scintillation counting analysisAgonist activity at rat MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr by liquid scintillation counting analysis
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.
Agonist activity at human mu opioid receptor expressed in CHO cell membranes incubated for 1 hr by [35S]GTPgammaS binding assayAgonist activity at human mu opioid receptor expressed in CHO cell membranes incubated for 1 hr by [35S]GTPgammaS binding assay
Agonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS bindingAgonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding
Agonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS bindingAgonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding
Agonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS bindingAgonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS binding
Agonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS bindingAgonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS binding
Activity at monocloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayActivity at monocloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at mouse mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding after 1.5 hrsAgonist activity at mouse mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding after 1.5 hrs
Stimulation of mouse MOR expressed in CHO cells after 1.5 hrs by [35S]GTPgammaS binding assayStimulation of mouse MOR expressed in CHO cells after 1.5 hrs by [35S]GTPgammaS binding assay
Activity at mu opioid receptor assessed as increase in calcium level in CHO cells by aequorin luminescence based calcium assayActivity at mu opioid receptor assessed as increase in calcium level in CHO cells by aequorin luminescence based calcium assay
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
Agonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS bindingAgonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding
Agonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS bindingAgonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding
Agonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS bindingAgonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS binding
Agonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS bindingAgonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS binding
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells after 2 hrs by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells after 2 hrs by [35S]GTPgammaS binding assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells after 2 hrs by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells after 2 hrs by [35S]GTPgammaS binding assay
Activity was evaluated in human mu opioid receptors transfected with CHO cells by [35S]GTP-gamma-S, assayActivity was evaluated in human mu opioid receptors transfected with CHO cells by [35S]GTP-gamma-S, assay
Agonist activity at human MOR expressed in CHOK1 cells assessed as inhibition of forskolin-stimulated cAMP accumulation incubated for 30 mins by luminescence assayAgonist activity at human MOR expressed in CHOK1 cells assessed as inhibition of forskolin-stimulated cAMP accumulation incubated for 30 mins by luminescence assay
Agonist activity at human mu opiod receptor expressed in CHO-K1 cells assessed as increase in forskolin induced cAMP production measured after 30 mins by HitHunter luminescence based assayAgonist activity at human mu opiod receptor expressed in CHO-K1 cells assessed as increase in forskolin induced cAMP production measured after 30 mins by HitHunter luminescence based assay
Agonist activity at human mu opioid receptor expressed in HEK293 cells by CellKey methodAgonist activity at human mu opioid receptor expressed in HEK293 cells by CellKey method
Agonist activity at human mu-opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation countingAgonist activity at human mu-opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation counting
Agonist activity at human recombinant MOP receptor expressed in CHO-K1 cells assessed as stimulation of [35S]GTPgammaS binding by scintillation proximity assayAgonist activity at human recombinant MOP receptor expressed in CHO-K1 cells assessed as stimulation of [35S]GTPgammaS binding by scintillation proximity assay
Agonist activity at human recombinant MOP receptor expressed in CHO-K1 cells assessed as stimulation of [35S]GTPgammaS binding by scintillation proximity assayAgonist activity at human recombinant MOP receptor expressed in CHO-K1 cells assessed as stimulation of [35S]GTPgammaS binding by scintillation proximity assay
Agonist activity at human recombinant MOP receptor expressed in CHO-K1 cells assessed as stimulation of [35S]GTPgammaS binding by scintillation proximity assayAgonist activity at human recombinant MOP receptor expressed in CHO-K1 cells assessed as stimulation of [35S]GTPgammaS binding by scintillation proximity assay
Agonist activity at human recombinant MOP receptor expressed in CHO-K1 cells assessed as stimulation of [35S]GTPgammaS binding by scintillation proximity assayAgonist activity at human recombinant MOP receptor expressed in CHO-K1 cells assessed as stimulation of [35S]GTPgammaS binding by scintillation proximity assay
Agonist activity at rat mu opioid receptor expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr by liquid scintillation countingAgonist activity at rat mu opioid receptor expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr by liquid scintillation counting
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Agonist activity at recombinant human mu opioid receptor expressed in CHOK1 cell membranes after 45 mins by GTPgamma(35)S binding assayAgonist activity at recombinant human mu opioid receptor expressed in CHOK1 cell membranes after 45 mins by GTPgamma(35)S binding assay
Agonist activity at recombinant human mu opioid receptor expressed in CHOK1 cell membranes after 45 mins by GTPgamma(35)S binding assayAgonist activity at recombinant human mu opioid receptor expressed in CHOK1 cell membranes after 45 mins by GTPgamma(35)S binding assay
Antagonist activity at mouse MOR expressed in CHO cell membranes assessed as inhibition of DAMGO-stimulated [35S]GTPgammaS binding after 90 minsAntagonist activity at mouse MOR expressed in CHO cell membranes assessed as inhibition of DAMGO-stimulated [35S]GTPgammaS binding after 90 mins
Inverse agonist activity at human recombinant mu opioid receptor expressed in CHO cells after 3 hrs by [35S]GTPgammaS binding assayInverse agonist activity at human recombinant mu opioid receptor expressed in CHO cells after 3 hrs by [35S]GTPgammaS binding assay
Inverse agonist activity at human recombinant mu opioid receptor expressed in CHO cells after 3 hrs by [35S]GTPgammaS binding assayInverse agonist activity at human recombinant mu opioid receptor expressed in CHO cells after 3 hrs by [35S]GTPgammaS binding assay
Agonist activity at mouse mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding after 1.5 hrsAgonist activity at mouse mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding after 1.5 hrs
Stimulation of mouse MOR expressed in CHO cells after 1.5 hrs by [35S]GTPgammaS binding assayStimulation of mouse MOR expressed in CHO cells after 1.5 hrs by [35S]GTPgammaS binding assay
Activity at monocloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayActivity at monocloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human mu opioid receptor expressed in CHO cell membranes incubated for 1 hr by [35S]GTPgammaS binding assayAgonist activity at human mu opioid receptor expressed in CHO cell membranes incubated for 1 hr by [35S]GTPgammaS binding assay
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Antagonist activity at mouse MOR expressed in CHO cell membranes assessed as inhibition of DAMGO-stimulated [35S]GTPgammaS binding after 90 minsAntagonist activity at mouse MOR expressed in CHO cell membranes assessed as inhibition of DAMGO-stimulated [35S]GTPgammaS binding after 90 mins
Activity at rat mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayActivity at rat mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human mu opioid receptor expressed in CHO cell membranes after 1 hr by [35S]-GTPgammaS binding assayAgonist activity at human mu opioid receptor expressed in CHO cell membranes after 1 hr by [35S]-GTPgammaS binding assay
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as maximal stimulation of [35S]GTP-gamma-S bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as maximal stimulation of [35S]GTP-gamma-S binding
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at human mu opioid receptor expressed in CHO-K1 cells assessed as inhibition of forskolin-induced cAMP accumulation incubated for 30 mins by luminescence-based assayAgonist activity at human mu opioid receptor expressed in CHO-K1 cells assessed as inhibition of forskolin-induced cAMP accumulation incubated for 30 mins by luminescence-based assay
Agonist activity at mu opioid receptor (unknown origin) expressed in CHO cellsAgonist activity at mu opioid receptor (unknown origin) expressed in CHO cells
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membrane assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in rat C6 cell membrane assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.
Inhibitory activity in stimulating [35S]-GTP-gamma S binding mediated by the Opioid receptor mu 1 in chinese Hamster Ovary (CHO) cell membranes was determinedInhibitory activity in stimulating [35S]-GTP-gamma S binding mediated by the Opioid receptor mu 1 in chinese Hamster Ovary (CHO) cell membranes was determined
Partial agonist activity at human mu opioid receptor expressed in CHO cells by [35S]GTPgammaS bindingPartial agonist activity at human mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding
Agonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS bindingAgonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding
Agonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS bindingAgonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding
Agonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS bindingAgonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS binding
Agonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS bindingAgonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS binding
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
Agonist activity at recombinant human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assayAgonist activity at recombinant human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assay
Agonist activity at mouse MOR expressed in CHO cell membranes assessed as induction of [35S]GTPgammaS binding after 90 mins by scintillation spectroscopyAgonist activity at mouse MOR expressed in CHO cell membranes assessed as induction of [35S]GTPgammaS binding after 90 mins by scintillation spectroscopy
Agonist activity at mouse cloned mu opioid receptor expressed in CHO cells after 60 mins by [35S]GTPgammaS binding assayAgonist activity at mouse cloned mu opioid receptor expressed in CHO cells after 60 mins by [35S]GTPgammaS binding assay
Antagonist activity at mouse MOR expressed in CHO cell membranes assessed as inhibition of DAMGO-stimulated [35S]GTPgammaS binding after 90 minsAntagonist activity at mouse MOR expressed in CHO cell membranes assessed as inhibition of DAMGO-stimulated [35S]GTPgammaS binding after 90 mins
Activity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cellsActivity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cells
Agonist activity at mouse mu opioid receptor-1 expressed in CHO cell membranes assessed as [35S]GTPgammaS binding incubated for 60 mins by scintillation spectroscopic analysisAgonist activity at mouse mu opioid receptor-1 expressed in CHO cell membranes assessed as [35S]GTPgammaS binding incubated for 60 mins by scintillation spectroscopic analysis
Agonist activity at mouse mu opioid receptor-1 expressed in CHO cell membranes assessed as [35S]GTPgammaS binding incubated for 60 mins by scintillation spectroscopic analysisAgonist activity at mouse mu opioid receptor-1 expressed in CHO cell membranes assessed as [35S]GTPgammaS binding incubated for 60 mins by scintillation spectroscopic analysis
Agonist activity at rat mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assayAgonist activity at rat mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assay
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding
Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.
Inhibition of mu opioid receptor (unknown origin) in presence of DAMGO by [35S]-GTPgammaS binding assayInhibition of mu opioid receptor (unknown origin) in presence of DAMGO by [35S]-GTPgammaS binding assay
Inhibition of mu opioid receptor (unknown origin) in presence of DPDPE by [35S]-GTPgammaS binding assayInhibition of mu opioid receptor (unknown origin) in presence of DPDPE by [35S]-GTPgammaS binding assay
μ-Opioid Receptor Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared in-house from a cell line expressing recombinant μ opioid receptor in a HEK293 background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25 °C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50 °C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.μ-Opioid Receptor Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared in-house from a cell line expressing recombinant μ opioid receptor in a HEK293 background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25 °C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50 °C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
Activity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cellsActivity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cells
Agonist activity at MOP (unknown origin) expressed in SH-SY5Y cell membranes co-expressing Gbeta1-RGFP protein assessed as induction of G protein activation incubated for 15 mins by BRET assayAgonist activity at MOP (unknown origin) expressed in SH-SY5Y cell membranes co-expressing Gbeta1-RGFP protein assessed as induction of G protein activation incubated for 15 mins by BRET assay
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
Agonist activity at recombinant human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assayAgonist activity at recombinant human MOR expressed in CHO cell membranes after 60 mins by [35S]GTPgammaS binding assay
Displacement of [3H]DAMGO from mu opioid receptor (unknown origin) expressed in HEK293 cells after 1 hr by liquid scintillation countingDisplacement of [3H]DAMGO from mu opioid receptor (unknown origin) expressed in HEK293 cells after 1 hr by liquid scintillation counting
Agonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS bindingAgonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding
Agonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS bindingAgonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding
Agonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS bindingAgonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS binding
Agonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS bindingAgonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS binding
Antagonist activity at mouse MOR expressed in CHO cell membranes assessed as inhibition of DAMGO-stimulated [35S]GTPgammaS binding after 90 minsAntagonist activity at mouse MOR expressed in CHO cell membranes assessed as inhibition of DAMGO-stimulated [35S]GTPgammaS binding after 90 mins
Agonist activity at human mu opioid receptor expressed in CHO cell membranes incubated for 1 hr by [35S]GTPgammaS binding assayAgonist activity at human mu opioid receptor expressed in CHO cell membranes incubated for 1 hr by [35S]GTPgammaS binding assay
Agonist activity at human mu opioid receptor expressed in human U2OS cells co-transfected with beta-arrestin-2 assessed as increase in beta-arrestin-2 recruitment after 90 mins by BRET assayAgonist activity at human mu opioid receptor expressed in human U2OS cells co-transfected with beta-arrestin-2 assessed as increase in beta-arrestin-2 recruitment after 90 mins by BRET assay
Agonist activity at mouse mu opioid receptor-1 expressed in CHO cell membranes assessed as [35S]GTPgammaS binding incubated for 60 mins by scintillation spectroscopic analysisAgonist activity at mouse mu opioid receptor-1 expressed in CHO cell membranes assessed as [35S]GTPgammaS binding incubated for 60 mins by scintillation spectroscopic analysis
Agonist activity at rat MOR transfected in rat C6 cell membranes after 1 hr by [35S]GTPgammaS assayAgonist activity at rat MOR transfected in rat C6 cell membranes after 1 hr by [35S]GTPgammaS assay
Agonist activity at rat mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assayAgonist activity at rat mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assay
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.
Partial agonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding after 60 mins by scintillation countingPartial agonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding after 60 mins by scintillation counting
Agonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS bindingAgonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding
Agonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS bindingAgonist activity against mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding
Agonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS bindingAgonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS binding
Agonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS bindingAgonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS binding
Agonist activity at human mu opioid receptor transfected in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by beta-plate liquid scintillation counting analysisAgonist activity at human mu opioid receptor transfected in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by beta-plate liquid scintillation counting analysis
μ-Opioid Receptor Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared in-house from a cell line expressing recombinant μ opioid receptor in a HEK293 background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25 °C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50 °C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.μ-Opioid Receptor Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared in-house from a cell line expressing recombinant μ opioid receptor in a HEK293 background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25 °C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50 °C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Activity at human mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assayActivity at human mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assay
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as maximal stimulation of [35S]GTP-gamma-S bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as maximal stimulation of [35S]GTP-gamma-S binding
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [33S]GTPgammaS binding after 60 mins by scintillation countingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [33S]GTPgammaS binding after 60 mins by scintillation counting
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding after 60 minsAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding after 60 mins
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at human opioid gamma receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S bindingAgonist activity at human opioid gamma receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding
Agonist activity at rat MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr by liquid scintillation counting analysisAgonist activity at rat MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr by liquid scintillation counting analysis
Agonist activity at rat MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr by liquid scintillation counting analysisAgonist activity at rat MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr by liquid scintillation counting analysis
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor transfected in HN9.10 cells by [35S]GTPgammaS binding assayAgonist activity at rat mu opioid receptor transfected in HN9.10 cells by [35S]GTPgammaS binding assay
Agonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysisAgonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysis
Agonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysisAgonist activity at rat mu-opioid receptor expressed in C6 cell membrane assessed as [35S]GTPgammaS binding for 1 hr by liquid scintillation counting analysis
Agonistic activity against Opioid receptor mu 1 in Chinese hamster ovary membranesAgonistic activity against Opioid receptor mu 1 in Chinese hamster ovary membranes
Partial agonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding after 60 mins by scintillation countingPartial agonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding after 60 mins by scintillation counting
Agonist activity at human mu opioid receptor expressed in CHO cell membranes incubated for 1 hr by [35S]GTPgammaS binding assayAgonist activity at human mu opioid receptor expressed in CHO cell membranes incubated for 1 hr by [35S]GTPgammaS binding assay
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
Agonist activity at human recombinant MOR expressed in CHO cells co-expressing C-terminally modified Galphaqi5 assessed as induction of calcium mobilization by Fluo-4AM dye-based fluorescence assayAgonist activity at human recombinant MOR expressed in CHO cells co-expressing C-terminally modified Galphaqi5 assessed as induction of calcium mobilization by Fluo-4AM dye-based fluorescence assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells co-expressing C-terminally modified GGalphaqi5 assessed as stimulation of calcium release by Fluo-4 AM dye-based fluorescence assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells co-expressing C-terminally modified GGalphaqi5 assessed as stimulation of calcium release by Fluo-4 AM dye-based fluorescence assay
Agonist activity at mu opioid receptor (unknown origin) expressed CHO cells assessed as stimulation of [35S]-GTP[gammaS] bindingAgonist activity at mu opioid receptor (unknown origin) expressed CHO cells assessed as stimulation of [35S]-GTP[gammaS] binding
Agonist activity at mu opioid receptor (unknown origin) expressed CHO cells assessed as stimulation of [35S]-GTP[gammaS] bindingAgonist activity at mu opioid receptor (unknown origin) expressed CHO cells assessed as stimulation of [35S]-GTP[gammaS] binding
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at human mu opioid receptor expressed in CHO membrane assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at human mu opioid receptor expressed in CHO membrane assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at human mu-opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation countingAgonist activity at human mu-opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation counting
Agonist activity at mouse mu opioid receptor-1 expressed in CHO cell membranes assessed as [35S]GTPgammaS binding incubated for 60 mins by scintillation spectroscopic analysisAgonist activity at mouse mu opioid receptor-1 expressed in CHO cell membranes assessed as [35S]GTPgammaS binding incubated for 60 mins by scintillation spectroscopic analysis
Agonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hrAgonist activity at rat recombinant MOR expressed in rat C6 cells assessed as stimulation of [35S]GTPgammaS binding after 1 hr
Inhibition of mu opioid receptor (unknown origin) in presence of DAMGO by [35S]-GTPgammaS binding assayInhibition of mu opioid receptor (unknown origin) in presence of DAMGO by [35S]-GTPgammaS binding assay
Inhibition of mu opioid receptor (unknown origin) in presence of DPDPE by [35S]-GTPgammaS binding assayInhibition of mu opioid receptor (unknown origin) in presence of DPDPE by [35S]-GTPgammaS binding assay
Partial agonist activity at human mu opioid receptor expressed in CHO cell membrane assessed as stimulation of [35S]GTPgammaS binding after 60 mins by liquid scintillation countingPartial agonist activity at human mu opioid receptor expressed in CHO cell membrane assessed as stimulation of [35S]GTPgammaS binding after 60 mins by liquid scintillation counting
Activity at human MOR expressed in CHOK1 cells co-expressing beta-arrestin2 EFC assessed as beta-arrestin2 EFC recruitment measured after 90 mins by PathHunter assayActivity at human MOR expressed in CHOK1 cells co-expressing beta-arrestin2 EFC assessed as beta-arrestin2 EFC recruitment measured after 90 mins by PathHunter assay
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding by scintillation counting analysisAgonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding by scintillation counting analysis
Agonist activity at mouse mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding after 1.5 hrsAgonist activity at mouse mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTPgammaS binding after 1.5 hrs
Stimulation of mouse MOR expressed in CHO cells after 1.5 hrs by [35S]GTPgammaS binding assayStimulation of mouse MOR expressed in CHO cells after 1.5 hrs by [35S]GTPgammaS binding assay
Agonist activity at human delta opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S bindingAgonist activity at human delta opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding
Agonist activity at human mu opioid receptor expressed in HEK293 cells by CellKey methodAgonist activity at human mu opioid receptor expressed in HEK293 cells by CellKey method
Agonist activity at human mu-opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation countingAgonist activity at human mu-opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation counting
Agonist activity at rat MOR transfected in rat C6 cell membranes after 1 hr by [35S]GTPgammaS assayAgonist activity at rat MOR transfected in rat C6 cell membranes after 1 hr by [35S]GTPgammaS assay
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membrane assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in rat C6 cell membrane assessed as stimulation of [35S]GTPgammaS binding
EC50 for binding of [35S]- GTPdeltaS in cloned human Opioid receptor mu 1 (DAMGO) transfected onto CHO cellsEC50 for binding of [35S]- GTPdeltaS in cloned human Opioid receptor mu 1 (DAMGO) transfected onto CHO cells
[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.
Antagonist activity at mouse MOR expressed in CHO cell membranes assessed as inhibition of DAMGO-stimulated [35S]GTPgammaS binding after 90 minsAntagonist activity at mouse MOR expressed in CHO cell membranes assessed as inhibition of DAMGO-stimulated [35S]GTPgammaS binding after 90 mins
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Agonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting methodAgonist activity at recombinant mouse mu opioid receptor expressed in CHO cell membrane assessed as [35]-GTPgammaS binding incubated for 1.5 hrs by scintillation counting method
Antagonist activity at mouse MOR expressed in CHO cell membranes assessed as inhibition of DAMGO-stimulated [35S]GTPgammaS binding after 90 minsAntagonist activity at mouse MOR expressed in CHO cell membranes assessed as inhibition of DAMGO-stimulated [35S]GTPgammaS binding after 90 mins
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
Agonist activity at human mu opioid receptor expressed in CHO cell membranes incubated for 1 hr by [35S]GTPgammaS binding assayAgonist activity at human mu opioid receptor expressed in CHO cell membranes incubated for 1 hr by [35S]GTPgammaS binding assay
Activity at rat mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayActivity at rat mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Activity was evaluated in human mu opioid receptors transfected with CHO cells by [35S]GTP-gamma-S, assayActivity was evaluated in human mu opioid receptors transfected with CHO cells by [35S]GTP-gamma-S, assay
Agonist activity at human MOR expressed in CHO cell membranes assessed as stimulation of [35S]-GTPgammaS binding incubated for 1 hr by by liquid scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes assessed as stimulation of [35S]-GTPgammaS binding incubated for 1 hr by by liquid scintillation counting assay
Agonist activity at human MOR expressed in CHO cell membranes assessed as stimulation of [35S]-GTPgammaS binding incubated for 1 hr by by liquid scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes assessed as stimulation of [35S]-GTPgammaS binding incubated for 1 hr by by liquid scintillation counting assay
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting methodAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting method
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting methodAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting method
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Concentration necessary to produce 50% of the Emax value, i.e. to stimulate [35S]GTP-gamma-S, binding to recombinant human Opioid receptor mu 1 expressed in CHO cellsConcentration necessary to produce 50% of the Emax value, i.e. to stimulate [35S]GTP-gamma-S, binding to recombinant human Opioid receptor mu 1 expressed in CHO cells
Agonist activity at mu opioid receptor in Swiss albino mouse ileum assessed as inhibition of electrically-stimulated muscle contractionAgonist activity at mu opioid receptor in Swiss albino mouse ileum assessed as inhibition of electrically-stimulated muscle contraction
Activity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cellsActivity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cells
Agonist activity at human mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation by fluorescence assayAgonist activity at human mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation by fluorescence assay
Agonist activity at human mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation by fluorescence assayAgonist activity at human mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation by fluorescence assay
Agonist activity at human MOR expressed in CHO-K1 cells co-expressing Galpha 15 assessed as increase in intracellular calcium release incubated for 1 measured every 1.52 secs for 90 secs by FLIPR assayAgonist activity at human MOR expressed in CHO-K1 cells co-expressing Galpha 15 assessed as increase in intracellular calcium release incubated for 1 measured every 1.52 secs for 90 secs by FLIPR assay
Agonist activity at human MOR expressed in CHO-K1 cells co-expressing Galpha 15 assessed as increase in intracellular calcium release incubated for 1 measured every 1.52 secs for 90 secs by FLIPR assayAgonist activity at human MOR expressed in CHO-K1 cells co-expressing Galpha 15 assessed as increase in intracellular calcium release incubated for 1 measured every 1.52 secs for 90 secs by FLIPR assay
Agonist activity at human MOR expressed in CHO-K1 cells co-expressing Galpha 15 assessed as increase in intracellular calcium release incubated for 1 measured every 1.52 secs for 90 secs by FLIPR assayAgonist activity at human MOR expressed in CHO-K1 cells co-expressing Galpha 15 assessed as increase in intracellular calcium release incubated for 1 measured every 1.52 secs for 90 secs by FLIPR assay
Agonist activity at human MOR expressed in CHOK1 cells assessed as stimulation of cAMP accumulation incubated for 45 mins by HTRF assayAgonist activity at human MOR expressed in CHOK1 cells assessed as stimulation of cAMP accumulation incubated for 45 mins by HTRF assay
Agonist activity at human mu opioid receptor expressed CHO-K1 cells coexpressing Galpha15 assessed as induction of intracellular calcium release by FLIPR calcium assayAgonist activity at human mu opioid receptor expressed CHO-K1 cells coexpressing Galpha15 assessed as induction of intracellular calcium release by FLIPR calcium assay
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at human mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation by fluorescence assayAgonist activity at human mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation by fluorescence assay
Agonist activity at human mu-opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation countingAgonist activity at human mu-opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation counting
Agonist activity at human recombinant MOR expressed in CHO cells assessed as cAMP accumulationAgonist activity at human recombinant MOR expressed in CHO cells assessed as cAMP accumulation
Agonist activity at mouse MOR assessed as inhibition of forskolin-stimulated cAMP accumulation after 30 mins by cell based TR-FRET assayAgonist activity at mouse MOR assessed as inhibition of forskolin-stimulated cAMP accumulation after 30 mins by cell based TR-FRET assay
Agonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS bindingAgonist activity at mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]-GTP-gammaS binding
Agonist activity at rat MOR expressed in HN9.10 cell membranes by [35S]GTPgammaS binding assayAgonist activity at rat MOR expressed in HN9.10 cell membranes by [35S]GTPgammaS binding assay
Inhibition of [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in CHO cellsInhibition of [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in CHO cells
Inhibitory activity in stimulating [35S]-GTP-gamma S binding mediated by the Opioid receptor mu 1 in chinese Hamster Ovary (CHO) cell membranes was determinedInhibitory activity in stimulating [35S]-GTP-gamma S binding mediated by the Opioid receptor mu 1 in chinese Hamster Ovary (CHO) cell membranes was determined
The binding affinity was evaluated against [3H]dalamid binding to rat brain membranes (mu opiate receptors).The binding affinity was evaluated against [3H]dalamid binding to rat brain membranes (mu opiate receptors).
The binding affinity was evaluated against [3H]dalamid binding to rat brain membranes (mu opiate receptors).The binding affinity was evaluated against [3H]dalamid binding to rat brain membranes (mu opiate receptors).
Agonist activity at human mu opioid receptor transfected in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by beta-plate liquid scintillation counting analysisAgonist activity at human mu opioid receptor transfected in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by beta-plate liquid scintillation counting analysis
Agonist activity at human mu opioid receptor transfected in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by beta-plate liquid scintillation counting analysisAgonist activity at human mu opioid receptor transfected in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by beta-plate liquid scintillation counting analysis
Partial agonist activity at human MOP receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation countingPartial agonist activity at human MOP receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation counting
Partial agonist activity at human MOP receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation countingPartial agonist activity at human MOP receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by scintillation counting
Agonist activity at human recombinant MOR expressed in CHO cells assessed as calcium mobilization by Fluo-4 AM based fluorescence analysisAgonist activity at human recombinant MOR expressed in CHO cells assessed as calcium mobilization by Fluo-4 AM based fluorescence analysis
[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.
Agonist activity at human mu opioid receptor transfected in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by beta-plate liquid scintillation counting analysisAgonist activity at human mu opioid receptor transfected in CHO cells assessed as stimulation of [35S]GTPgammaS binding after 60 mins by beta-plate liquid scintillation counting analysis
Agonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu-opioid receptor expressed in CHO cells after 90 mins by [35S]GTP-gamma-S assayAgonist activity at rat mu-opioid receptor expressed in CHO cells after 90 mins by [35S]GTP-gamma-S assay
Antagonist activity at human recombinant mu opioid receptor expressed in CHO cells coexpressing alphaqi5 assessed as inhibition of dermorphin-induced intracellular calcium mobilizationAntagonist activity at human recombinant mu opioid receptor expressed in CHO cells coexpressing alphaqi5 assessed as inhibition of dermorphin-induced intracellular calcium mobilization
Inhibition of [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in CHO cellsInhibition of [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in CHO cells
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding
Agonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counterAgonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counter
Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.
Agonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counterAgonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counter
Agonist activity at MOP (unknown origin) expressed in SH-SY5Y cell membranes co-expressing beta-arrestin2-RGFP protein assessed as induction of beta-arrestin2 activation incubated for 15 mins by BRET assayAgonist activity at MOP (unknown origin) expressed in SH-SY5Y cell membranes co-expressing beta-arrestin2-RGFP protein assessed as induction of beta-arrestin2 activation incubated for 15 mins by BRET assay
Agonist activity at rat mu-opioid receptor expressed in CHO cells after 90 mins by [35S]GTP-gamma-S assayAgonist activity at rat mu-opioid receptor expressed in CHO cells after 90 mins by [35S]GTP-gamma-S assay
Antagonist activity at human recombinant mu opioid receptor expressed in CHO cells coexpressing alphaqi5 assessed as inhibition of dermorphin-induced intracellular calcium mobilizationAntagonist activity at human recombinant mu opioid receptor expressed in CHO cells coexpressing alphaqi5 assessed as inhibition of dermorphin-induced intracellular calcium mobilization
Agonist activity at human mu opioid receptor expressed CHO-K1 cells coexpressing Galpha15 assessed as induction of intracellular calcium release by FLIPR calcium assayAgonist activity at human mu opioid receptor expressed CHO-K1 cells coexpressing Galpha15 assessed as induction of intracellular calcium release by FLIPR calcium assay
Agonist activity at myc-tagged mu opioid receptor (unknown origin) expressed in AtT-20 cells assessed as induction of membrane potential hyperpolarizationAgonist activity at myc-tagged mu opioid receptor (unknown origin) expressed in AtT-20 cells assessed as induction of membrane potential hyperpolarization
Agonist activity at rat MOR expressed in HN9.10 cell membranes by [35S]GTPgammaS binding assayAgonist activity at rat MOR expressed in HN9.10 cell membranes by [35S]GTPgammaS binding assay
Agonist activity at rat mu opioid receptor transfected in HN9.10 cells by [35S]GTPgammaS binding assayAgonist activity at rat mu opioid receptor transfected in HN9.10 cells by [35S]GTPgammaS binding assay
Agonist activity at human MOR expressed in CHO cell membranes after 60 mins by [35S]GTP-gamma-S binding assayAgonist activity at human MOR expressed in CHO cell membranes after 60 mins by [35S]GTP-gamma-S binding assay
Agonist activity at human MOR expressed in CHO cell membranes after 60 mins by [35S]GTP-gamma-S binding assayAgonist activity at human MOR expressed in CHO cell membranes after 60 mins by [35S]GTP-gamma-S binding assay
Agonist activity at human mu opioid receptor expressed in CHO-K1 cells assessed as stimulation of [35S]-GTPgammaS binding by scintillation proximity assayAgonist activity at human mu opioid receptor expressed in CHO-K1 cells assessed as stimulation of [35S]-GTPgammaS binding by scintillation proximity assay
Agonist activity at human mu opioid receptor expressed in CHO-K1 cells assessed as stimulation of [35S]-GTPgammaS binding by scintillation proximity assayAgonist activity at human mu opioid receptor expressed in CHO-K1 cells assessed as stimulation of [35S]-GTPgammaS binding by scintillation proximity assay
Agonist activity at human mu opioid receptor expressed in CHO-K1 cells assessed as stimulation of [35S]-GTPgammaS binding by scintillation proximity assayAgonist activity at human mu opioid receptor expressed in CHO-K1 cells assessed as stimulation of [35S]-GTPgammaS binding by scintillation proximity assay
Agonist activity at human mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation by fluorescence assayAgonist activity at human mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation by fluorescence assay
Agonist activity at human MOR expressed in CHO-K1 cells co-expressing Galpha 15 assessed as increase in intracellular calcium release incubated for 1 measured every 1.52 secs for 90 secs by FLIPR assayAgonist activity at human MOR expressed in CHO-K1 cells co-expressing Galpha 15 assessed as increase in intracellular calcium release incubated for 1 measured every 1.52 secs for 90 secs by FLIPR assay
Agonist activity at human mu opioid receptor expressed CHO-K1 cells coexpressing Galpha15 assessed as induction of intracellular calcium release by FLIPR calcium assayAgonist activity at human mu opioid receptor expressed CHO-K1 cells coexpressing Galpha15 assessed as induction of intracellular calcium release by FLIPR calcium assay
Agonist activity at human mu opioid receptor expressed CHO-K1 cells coexpressing Galpha15 assessed as induction of intracellular calcium release by FLIPR calcium assayAgonist activity at human mu opioid receptor expressed CHO-K1 cells coexpressing Galpha15 assessed as induction of intracellular calcium release by FLIPR calcium assay
Agonist activity at human mu opioid receptor expressed in CHOK1 cells by [35S]GTPgammaS bindingAgonist activity at human mu opioid receptor expressed in CHOK1 cells by [35S]GTPgammaS binding
Agonist activity at human mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation by fluorescence assayAgonist activity at human mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation by fluorescence assay
Agonist activity at human mu-type opioid receptor expressed in CHO-K1 cells assessed as cAMP accumulation by HTRF assayAgonist activity at human mu-type opioid receptor expressed in CHO-K1 cells assessed as cAMP accumulation by HTRF assay
Positive allosteric modulation of human mu opioid receptor-1 expressed in CHO cells assessed as potentiation of endomorphin 1-induced response after 90 mins by beta-arrestin recruitment assayPositive allosteric modulation of human mu opioid receptor-1 expressed in CHO cells assessed as potentiation of endomorphin 1-induced response after 90 mins by beta-arrestin recruitment assay
Agonist activity at human mu opioid receptor expressed in COS7 cells after 60 mins IP1 assayAgonist activity at human mu opioid receptor expressed in COS7 cells after 60 mins IP1 assay
Agonist activity at human mu opioid receptor expressed in COS7 cells after 60 mins IP1 assayAgonist activity at human mu opioid receptor expressed in COS7 cells after 60 mins IP1 assay
[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.
PubChem BioAssay. Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization. (Class of assay: confirmatory) PubChem BioAssay. Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization. (Class of assay: confirmatory)
Activity at human cloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayActivity at human cloned mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human mu opioid receptor expressed in CHOK1 cells coexpressing Galpha15 assessed as increase in intracellular calcium flux in presence of mu opioid receptor antagonist naloxone and incubated for 1 hr measured for 90 secs at 1.5 sec interval by FLIPR assayAgonist activity at human mu opioid receptor expressed in CHOK1 cells coexpressing Galpha15 assessed as increase in intracellular calcium flux in presence of mu opioid receptor antagonist naloxone and incubated for 1 hr measured for 90 secs at 1.5 sec interval by FLIPR assay
PubChem BioAssay. Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization. (Class of assay: confirmatory) PubChem BioAssay. Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization. (Class of assay: confirmatory)
PubChem BioAssay. Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization. (Class of assay: confirmatory) PubChem BioAssay. Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization. (Class of assay: confirmatory)
Agonist activity at human mu opioid receptor expressed in CHO cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by scintillation counting methodAgonist activity at human mu opioid receptor expressed in CHO cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by scintillation counting method
Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.
Agonist activity at human MOR expressed in CHO cell membranes assessed as stimulation of [35S]-GTPgammaS binding incubated for 1 hr by by liquid scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes assessed as stimulation of [35S]-GTPgammaS binding incubated for 1 hr by by liquid scintillation counting assay
Agonist activity at human MOR expressed in CHO cell membranes assessed as stimulation of [35S]-GTPgammaS binding incubated for 1 hr by by liquid scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes assessed as stimulation of [35S]-GTPgammaS binding incubated for 1 hr by by liquid scintillation counting assay
Activity at Wistar rat mu opioid receptor assessed as inhibition of low-frequency electrically-stimulated vas deferens contractionActivity at Wistar rat mu opioid receptor assessed as inhibition of low-frequency electrically-stimulated vas deferens contraction
Agonist activity at mu opioid receptor in Wistar rat brain membranes after 60 mins by [35S]GTPgammaS binding assayAgonist activity at mu opioid receptor in Wistar rat brain membranes after 60 mins by [35S]GTPgammaS binding assay
PubChem BioAssay. Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization. (Class of assay: confirmatory) PubChem BioAssay. Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization. (Class of assay: confirmatory)
Agonist activity at human mu opioid receptor expressed CHO-K1 cells coexpressing Galpha15 assessed as induction of intracellular calcium release by FLIPR calcium assayAgonist activity at human mu opioid receptor expressed CHO-K1 cells coexpressing Galpha15 assessed as induction of intracellular calcium release by FLIPR calcium assay
[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.
Activity at mu opioid receptor in Wistar rat brain assessed as stimulation of [35S]GTPgammaS bindingActivity at mu opioid receptor in Wistar rat brain assessed as stimulation of [35S]GTPgammaS binding
HiRange Homogenous Time-Resolved Fluorescence (HTRF) Assay: Briefly, suspensions of cells expressing either the mu, kappa or delta opioid receptors were prepared in buffer containing 0.5 mM isobutyl-methyl xanthine (IBMX). Cells were incubated with varying concentrations of PEG-opioid conjugates and 3 uM forskolin for 30 minutes at room temperature. cAMP was detected following a two-step assay protocol per the manufacturer's instructions and time resolved fluorescence was measured with the following settings: 330 nm excitation; 620 nm and 665 nm emission; 380 nm dichroic mirror. The 665 nm/620 nm ratio is expressed as Delta F % and test compound-related data is expressed as a percentage of average maximum response in wells without forskolin. EC50 values were calculated for each compound from a sigmoidal dose-response plot of concentrations versus maximum response.HiRange Homogenous Time-Resolved Fluorescence (HTRF) Assay: Briefly, suspensions of cells expressing either the mu, kappa or delta opioid receptors were prepared in buffer containing 0.5 mM isobutyl-methyl xanthine (IBMX). Cells were incubated with varying concentrations of PEG-opioid conjugates and 3 uM forskolin for 30 minutes at room temperature. cAMP was detected following a two-step assay protocol per the manufacturer's instructions and time resolved fluorescence was measured with the following settings: 330 nm excitation; 620 nm and 665 nm emission; 380 nm dichroic mirror. The 665 nm/620 nm ratio is expressed as Delta F % and test compound-related data is expressed as a percentage of average maximum response in wells without forskolin. EC50 values were calculated for each compound from a sigmoidal dose-response plot of concentrations versus maximum response.
Agonist activity at human MOP expressed in CHO cell membrane incubated for 60 mins by [35S]GTPgammaS binding assayAgonist activity at human MOP expressed in CHO cell membrane incubated for 60 mins by [35S]GTPgammaS binding assay
Agonist activity at human delta opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S bindingAgonist activity at human delta opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding
Agonist activity at human mu opioid receptor expressed in CHO cell membranes after 1 hr by [35S]-GTPgammaS binding assayAgonist activity at human mu opioid receptor expressed in CHO cell membranes after 1 hr by [35S]-GTPgammaS binding assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells after 2 hrs by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells after 2 hrs by [35S]GTPgammaS binding assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells after 2 hrs by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells after 2 hrs by [35S]GTPgammaS binding assay
HiRange Homogenous Time-Resolved Fluorescence (HTRF) Assay: Briefly, suspensions of cells expressing either the mu, kappa or delta opioid receptors were prepared in buffer containing 0.5 mM isobutyl-methyl xanthine (IBMX). Cells were incubated with varying concentrations of PEG-opioid conjugates and 3 uM forskolin for 30 minutes at room temperature. cAMP was detected following a two-step assay protocol per the manufacturer's instructions and time resolved fluorescence was measured with the following settings: 330 nm excitation; 620 nm and 665 nm emission; 380 nm dichroic mirror. The 665 nm/620 nm ratio is expressed as Delta F % and test compound-related data is expressed as a percentage of average maximum response in wells without forskolin. EC50 values were calculated for each compound from a sigmoidal dose-response plot of concentrations versus maximum response.HiRange Homogenous Time-Resolved Fluorescence (HTRF) Assay: Briefly, suspensions of cells expressing either the mu, kappa or delta opioid receptors were prepared in buffer containing 0.5 mM isobutyl-methyl xanthine (IBMX). Cells were incubated with varying concentrations of PEG-opioid conjugates and 3 uM forskolin for 30 minutes at room temperature. cAMP was detected following a two-step assay protocol per the manufacturer's instructions and time resolved fluorescence was measured with the following settings: 330 nm excitation; 620 nm and 665 nm emission; 380 nm dichroic mirror. The 665 nm/620 nm ratio is expressed as Delta F % and test compound-related data is expressed as a percentage of average maximum response in wells without forskolin. EC50 values were calculated for each compound from a sigmoidal dose-response plot of concentrations versus maximum response.
Agonist activity at human MOR expressed in human U2OS cells assessed as increase in beta arrestin2 recruitment incubated for 90 mins by pathhunter beta-arrestin assayAgonist activity at human MOR expressed in human U2OS cells assessed as increase in beta arrestin2 recruitment incubated for 90 mins by pathhunter beta-arrestin assay
Agonist activity at human MOR expressed in CHOK1 cells assessed as stimulation of cAMP accumulation incubated for 45 mins by HTRF assayAgonist activity at human MOR expressed in CHOK1 cells assessed as stimulation of cAMP accumulation incubated for 45 mins by HTRF assay
Agonist activity at rat Mu-type opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting methodAgonist activity at rat Mu-type opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting method
Agonist activity at rat Mu-type opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting methodAgonist activity at rat Mu-type opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting method
Agonist activity at mu opioid receptor in rat brain membranes assessed as stimulation of [35S]GTPgammaS binding after 60 mins in presence of GDPAgonist activity at mu opioid receptor in rat brain membranes assessed as stimulation of [35S]GTPgammaS binding after 60 mins in presence of GDP
Agonist activity at human mu opioid receptor expressed in human USOS-beta-arrestin-hMOR-PathHunter cells incubated for 90 mins by beta-arrestin-2 enzyme fragment complementation assayAgonist activity at human mu opioid receptor expressed in human USOS-beta-arrestin-hMOR-PathHunter cells incubated for 90 mins by beta-arrestin-2 enzyme fragment complementation assay
PubChem BioAssay. Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization. (Class of assay: confirmatory) PubChem BioAssay. Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization. (Class of assay: confirmatory)
Agonist activity at human MOR expressed in CHOK1 cells assessed as stimulation of cAMP accumulation incubated for 45 mins by HTRF assayAgonist activity at human MOR expressed in CHOK1 cells assessed as stimulation of cAMP accumulation incubated for 45 mins by HTRF assay
Activity at mu opioid receptor assessed as stimulation of [35S]GTP-gamma-S bindingActivity at mu opioid receptor assessed as stimulation of [35S]GTP-gamma-S binding
Activity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cellsActivity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cells
PubChem BioAssay. Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization. (Class of assay: confirmatory) PubChem BioAssay. Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization. (Class of assay: confirmatory)
Agonist activity at human MOR expressed in CHOK1 cells assessed as stimulation of cAMP accumulation incubated for 45 mins by HTRF assayAgonist activity at human MOR expressed in CHOK1 cells assessed as stimulation of cAMP accumulation incubated for 45 mins by HTRF assay
Agonist activity at human mu opiod receptor expressed in CHO-K1 cells assessed as increase in forskolin induced cAMP production measured after 30 mins by HitHunter luminescence based assayAgonist activity at human mu opiod receptor expressed in CHO-K1 cells assessed as increase in forskolin induced cAMP production measured after 30 mins by HitHunter luminescence based assay
Agonist activity at human mu opioid receptor expressed in CHO cell membrane by [35S]GTPgammaS binding assayAgonist activity at human mu opioid receptor expressed in CHO cell membrane by [35S]GTPgammaS binding assay
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as maximal stimulation of [35S]GTP-gamma-S bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as maximal stimulation of [35S]GTP-gamma-S binding
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu-opioid receptor expressed in CHO cells after 90 mins by [35S]GTP-gamma-S assayAgonist activity at rat mu-opioid receptor expressed in CHO cells after 90 mins by [35S]GTP-gamma-S assay
Inhibitory activity in stimulating [35S]-GTP-gamma S binding mediated by the Opioid receptor mu 1 in chinese Hamster Ovary (CHO) cell membranes was determinedInhibitory activity in stimulating [35S]-GTP-gamma S binding mediated by the Opioid receptor mu 1 in chinese Hamster Ovary (CHO) cell membranes was determined
Agonist activity at mu opioid receptor (unknown origin) expressed CHO cells assessed as stimulation of [35S]-GTP[gammaS] bindingAgonist activity at mu opioid receptor (unknown origin) expressed CHO cells assessed as stimulation of [35S]-GTP[gammaS] binding
Agonist activity at mu opioid receptor (unknown origin) expressed CHO cells assessed as stimulation of [35S]-GTP[gammaS] bindingAgonist activity at mu opioid receptor (unknown origin) expressed CHO cells assessed as stimulation of [35S]-GTP[gammaS] binding
Agonist activity at human delta opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S bindingAgonist activity at human delta opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding
Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.
Stimulation of mouse MOR expressed in CHO cells after 1.5 hrs by [35S]GTPgammaS binding assayStimulation of mouse MOR expressed in CHO cells after 1.5 hrs by [35S]GTPgammaS binding assay
Agonist activity at human recombinant MOR expressed in CHO cells assessed as calcium mobilization by Fluo-4 AM based fluorescence analysisAgonist activity at human recombinant MOR expressed in CHO cells assessed as calcium mobilization by Fluo-4 AM based fluorescence analysis
Activity at human MOR expressed in CHOK1 cells co-expressing beta-arrestin2 EFC assessed as beta-arrestin2 EFC recruitment measured after 90 mins by PathHunter assayActivity at human MOR expressed in CHOK1 cells co-expressing beta-arrestin2 EFC assessed as beta-arrestin2 EFC recruitment measured after 90 mins by PathHunter assay
Agonist activity at human mu opioid receptor expressed in CHO cell membranes by [35S]GTPgammaS binding assayAgonist activity at human mu opioid receptor expressed in CHO cell membranes by [35S]GTPgammaS binding assay
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding
Activity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cellsActivity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cells
Agonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counterAgonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counter
Agonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counterAgonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counter
Agonist activity at mu opioid receptor in Swiss albino mouse ileum assessed as inhibition of electrically-stimulated muscle contractionAgonist activity at mu opioid receptor in Swiss albino mouse ileum assessed as inhibition of electrically-stimulated muscle contraction
Activity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cellsActivity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cells
Activity at human mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assayActivity at human mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assay
Activity at human mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assayActivity at human mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assay
Agonist activity at EA-fragment beta-arrestin-tagged human MOR expressed in human U2OS cell membranes assessed as stimulation of [35S]-GTPgammaS binding after 60 mins by beta-galactosidase based scintillation counting methodAgonist activity at EA-fragment beta-arrestin-tagged human MOR expressed in human U2OS cell membranes assessed as stimulation of [35S]-GTPgammaS binding after 60 mins by beta-galactosidase based scintillation counting method
Agonist activity at human MOR expressed in CHO-K1 cells co-expressing Galpha 15 assessed as increase in intracellular calcium release incubated for 1 measured every 1.52 secs for 90 secs by FLIPR assayAgonist activity at human MOR expressed in CHO-K1 cells co-expressing Galpha 15 assessed as increase in intracellular calcium release incubated for 1 measured every 1.52 secs for 90 secs by FLIPR assay
Agonist activity at human mu opioid receptor expressed in CHO cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by scintillation counting methodAgonist activity at human mu opioid receptor expressed in CHO cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by scintillation counting method
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding by scintillation countingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding by scintillation counting
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding by scintillation countingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]GTPgammaS binding by scintillation counting
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as maximal stimulation of [35S]GTP-gamma-S bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as maximal stimulation of [35S]GTP-gamma-S binding
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as maximal stimulation of [35S]GTP-gamma-S bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as maximal stimulation of [35S]GTP-gamma-S binding
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding after 60 minsAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding after 60 mins
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding after 60 minsAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding after 60 mins
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at human mu opioid receptor expressed in CHO membrane assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at human mu opioid receptor expressed in CHO membrane assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at human mu opioid receptor expressed in CHO-dhfr(-) cells assessed as inhibition of forskolin-induced cAMP accumulation by TR-FRET assayAgonist activity at human mu opioid receptor expressed in CHO-dhfr(-) cells assessed as inhibition of forskolin-induced cAMP accumulation by TR-FRET assay
Agonist activity at human opioid gamma receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S bindingAgonist activity at human opioid gamma receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding
Agonist activity at human opioid gamma receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S bindingAgonist activity at human opioid gamma receptor expressed in CHO cells assessed as stimulation of [35S]GTP-gamma-S binding
Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.Functional Assay (GTPgammaS Binding): The EC50 and Imax for mu opioid receptors was determined using a [I35S]GTPgammaS binding assay. This assay measures the functional properties of a compound by quantifying the level of G-protein activation following agonist binding in studies using stably transfected cells, and is considered to be a measure of the efficacy of a compound. Membranes from CHO (Chinese Hamster Ovary) cells that stably expressed the cloned human Mu opioid receptor were used in the experiments. Specifically, in a final volume of 0.5 mL, 12 different concentrations of each test compound were incubated with 7.5 ug of CHO cell membranes that stably expressed the human mu opioid receptor. The assay buffer consisted of 50 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 0.2 mM EGTA, 3 uM GDP, and 100 mM NaCl. The final concentration of [35S]GTPgammaS was 0.080 nM. Nonspecific binding was measured by inclusion of 10 uM GTPgammaS. Binding was initiated by the addition of the membranes.
Agonistic activity against human opioid Mu receptor transfected into Chinese hamster ovary (CHO) cells using [3H]-DAMGO as radioligandAgonistic activity against human opioid Mu receptor transfected into Chinese hamster ovary (CHO) cells using [3H]-DAMGO as radioligand
Agonist activity at human mu opioid receptor expressed in CHO-dhfr(-) cells assessed as inhibition of forskolin-induced cAMP accumulation by TR-FRET assayAgonist activity at human mu opioid receptor expressed in CHO-dhfr(-) cells assessed as inhibition of forskolin-induced cAMP accumulation by TR-FRET assay
Inhibition of mu-opioid receptor (unknown origin) by [35S]-GTPgammaS binding assayInhibition of mu-opioid receptor (unknown origin) by [35S]-GTPgammaS binding assay
Agonist activity at human mu opioid receptor expressed in CHO cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by scintillation counting methodAgonist activity at human mu opioid receptor expressed in CHO cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by scintillation counting method
Agonist activity at rat Mu-type opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting methodAgonist activity at rat Mu-type opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting method
Agonist activity at rat Mu-type opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting methodAgonist activity at rat Mu-type opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting method
Agonist activity at MOR (unknown origin) expressed in HEK cells assessed as reduction in cAMP accumulation by split luciferase assayAgonist activity at MOR (unknown origin) expressed in HEK cells assessed as reduction in cAMP accumulation by split luciferase assay
Agonist activity at MOR (unknown origin) expressed in HEK cells assessed as reduction in cAMP accumulation by split luciferase assayAgonist activity at MOR (unknown origin) expressed in HEK cells assessed as reduction in cAMP accumulation by split luciferase assay
Agonist activity at human MOR expressed in CHO-K1 cells co-expressing Galpha 15 assessed as increase in intracellular calcium release incubated for 1 measured every 1.52 secs for 90 secs by FLIPR assayAgonist activity at human MOR expressed in CHO-K1 cells co-expressing Galpha 15 assessed as increase in intracellular calcium release incubated for 1 measured every 1.52 secs for 90 secs by FLIPR assay
Agonist activity at human MOR expressed in CHO-K1 cells co-expressing Galpha 15 assessed as increase in intracellular calcium release incubated for 1 measured every 1.52 secs for 90 secs by FLIPR assayAgonist activity at human MOR expressed in CHO-K1 cells co-expressing Galpha 15 assessed as increase in intracellular calcium release incubated for 1 measured every 1.52 secs for 90 secs by FLIPR assay
Agonist activity at human mu opioid receptor expressed CHO-K1 cells coexpressing Galpha15 assessed as induction of intracellular calcium release by FLIPR calcium assayAgonist activity at human mu opioid receptor expressed CHO-K1 cells coexpressing Galpha15 assessed as induction of intracellular calcium release by FLIPR calcium assay
Agonist activity at mu opioid receptor (unknown origin) expressed in human HEK293 cells assessed as increase in cAMP accumulation using [3H]-cAMP as substrate measured after 30mins by liquid scintillation counting methodAgonist activity at mu opioid receptor (unknown origin) expressed in human HEK293 cells assessed as increase in cAMP accumulation using [3H]-cAMP as substrate measured after 30mins by liquid scintillation counting method
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]-GTP[gammaS] bindingAgonist activity at human mu opioid receptor expressed in CHO cells assessed as [35S]-GTP[gammaS] binding
Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.
Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.
Agonist activity at human MOR expressed in human U2OS cells assessed as increase in beta arrestin2 recruitment incubated for 90 mins by pathhunter beta-arrestin assayAgonist activity at human MOR expressed in human U2OS cells assessed as increase in beta arrestin2 recruitment incubated for 90 mins by pathhunter beta-arrestin assay
Activity at Wistar rat mu opioid receptor assessed as inhibition of low-frequency electrically-stimulated vas deferens contraction in presence of naloxoneActivity at Wistar rat mu opioid receptor assessed as inhibition of low-frequency electrically-stimulated vas deferens contraction in presence of naloxone
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells after 2 hrs by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells after 2 hrs by [35S]GTPgammaS binding assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells after 2 hrs by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells after 2 hrs by [35S]GTPgammaS binding assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assay
[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.
[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.
Agonist activity at human mu opioid receptor expressed in CHO cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by scintillation counting methodAgonist activity at human mu opioid receptor expressed in CHO cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by scintillation counting method
In Vitro Mu-Opioid Receptor Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed membranes expressing human μ-receptors. Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; NEN). The prepared membrane solution (190 μL/well) was transferred to 96-shallow well polypropylene plates containing 10 μL of 20× concentrated stock solutions of the agonist DAMGO ([D-Ala2, N-methyl-Phe4 Gly-o15]-enkephalin) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25 °C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Packard, Meriden, Conn.) using a 96-well tissue harvester (Brandel, Gaithersburg, Md.) followed by three filtration washes with 200 μL of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50 °C. for 2-3 hr. BetaScint scintillation cocktail (Wallas, Turku, Finland) was added (50 μL/well) and plates were counted using a Packard Top-Count for 1 min/well.In Vitro Mu-Opioid Receptor Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed membranes expressing human μ-receptors. Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; NEN). The prepared membrane solution (190 μL/well) was transferred to 96-shallow well polypropylene plates containing 10 μL of 20× concentrated stock solutions of the agonist DAMGO ([D-Ala2, N-methyl-Phe4 Gly-o15]-enkephalin) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25 °C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Packard, Meriden, Conn.) using a 96-well tissue harvester (Brandel, Gaithersburg, Md.) followed by three filtration washes with 200 μL of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50 °C. for 2-3 hr. BetaScint scintillation cocktail (Wallas, Turku, Finland) was added (50 μL/well) and plates were counted using a Packard Top-Count for 1 min/well.
[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.
Agonist activity at human mu opioid receptor expressed in CHO cell membranes incubated for 1 hr by [35S]GTPgammaS binding assayAgonist activity at human mu opioid receptor expressed in CHO cell membranes incubated for 1 hr by [35S]GTPgammaS binding assay
Agonist activity at rat Mu-type opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting methodAgonist activity at rat Mu-type opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting method
Agonist activity at rat Mu-type opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting methodAgonist activity at rat Mu-type opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting method
Agonist activity at rat mu opioid receptor expressed in HN9.10 cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in HN9.10 cells assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at EA-fragment beta-arrestin-tagged human MOR expressed in human U2OS cell membranes assessed as stimulation beta-arrestin recruitment after 60 mins by Pathhunter chemiluminescence assayAgonist activity at EA-fragment beta-arrestin-tagged human MOR expressed in human U2OS cell membranes assessed as stimulation beta-arrestin recruitment after 60 mins by Pathhunter chemiluminescence assay
Agonist activity at rat mu opioid receptor expressed in rat C6 cells assessed as stimulation of [35S]-GTPgammaS binding after 1 hr by liquid scintillation counting analysisAgonist activity at rat mu opioid receptor expressed in rat C6 cells assessed as stimulation of [35S]-GTPgammaS binding after 1 hr by liquid scintillation counting analysis
Agonist activity at human mu opioid receptor transfected in CHO cells assessed as stimulation of [35S]GTPgammaS binding by liquid scintillation counting analysisAgonist activity at human mu opioid receptor transfected in CHO cells assessed as stimulation of [35S]GTPgammaS binding by liquid scintillation counting analysis
Activity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cellsActivity against [35S]GTP-gamma-S binding to rat mu opioid receptor expressed in HN9.10 cells
Activity at human mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assayActivity at human mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assay
Agonist activity at human mu opioid receptor expressed in CHO cell membrane by [35S]GTPgammaS binding assayAgonist activity at human mu opioid receptor expressed in CHO cell membrane by [35S]GTPgammaS binding assay
Agonist activity at human mu opioid receptor expressed in CHO membrane by [35S]GTP-gamma-S binding assayAgonist activity at human mu opioid receptor expressed in CHO membrane by [35S]GTP-gamma-S binding assay
Agonist activity at human mu-opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation preincubated for 15 mins followed by forskolin addition in presence of IBMX by Glo-sensor cAMP assayAgonist activity at human mu-opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation preincubated for 15 mins followed by forskolin addition in presence of IBMX by Glo-sensor cAMP assay
Agonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counterAgonist activity at mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-induced cAMP accumulation after 30 mins by liquid scintillation counter
Agonist activity at rat mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assayAgonist activity at rat mu opioid receptor expressed in CHO cells by [35S]GTP-gamma-S binding assay
Agonist activity at rat mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayAgonist activity at rat mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membrane assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in rat C6 cell membrane assessed as stimulation of [35S]GTPgammaS binding
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hrAgonist activity at rat mu opioid receptor expressed in rat C6 cell membranes assessed as stimulation of [35S]GTPgammaS binding measured after 1 hr
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.
Activity at human MOR expressed in U2OS cells co-expressing beta-arrestin2 EFC assessed as beta-arrestin2 EFC recruitment by Tango assayActivity at human MOR expressed in U2OS cells co-expressing beta-arrestin2 EFC assessed as beta-arrestin2 EFC recruitment by Tango assay
Agonist activity at Rluc-tagged MOR receptor (unknown origin) expressed in human SH-5YSY cells co-expressing RGFP-fused to Gbeta1 assessed as increase in MOR/G beta1 protein interaction incubated for 5 mins in presence of coelenterazine by BRET assayAgonist activity at Rluc-tagged MOR receptor (unknown origin) expressed in human SH-5YSY cells co-expressing RGFP-fused to Gbeta1 assessed as increase in MOR/G beta1 protein interaction incubated for 5 mins in presence of coelenterazine by BRET assay
Agonist activity at human mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation by fluorescence assayAgonist activity at human mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation by fluorescence assay
Agonist activity at human mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation by fluorescence assayAgonist activity at human mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation by fluorescence assay
Agonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assayAgonist activity at human MOR expressed in CHO cell membranes incubated for 60 mins scintillation counting assay
[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.
Agonist activity at human MOR stably expressed in CHO cell membranes assessed as stimulation of [35S]-GTPgammaS binding incubated for 1 hr by liquid scintillation counting assayAgonist activity at human MOR stably expressed in CHO cell membranes assessed as stimulation of [35S]-GTPgammaS binding incubated for 1 hr by liquid scintillation counting assay
Agonist activity at human MOR expressed in CHO-K1 cells co-expressing Galpha 15 assessed as increase in intracellular calcium release incubated for 1 measured every 1.52 secs for 90 secs by FLIPR assayAgonist activity at human MOR expressed in CHO-K1 cells co-expressing Galpha 15 assessed as increase in intracellular calcium release incubated for 1 measured every 1.52 secs for 90 secs by FLIPR assay
Agonist activity at human mu opioid receptor expressed in CHO cells assessed as inhibition of forskolin-induced cAMP accumulation after 1 hr by HTRF assayAgonist activity at human mu opioid receptor expressed in CHO cells assessed as inhibition of forskolin-induced cAMP accumulation after 1 hr by HTRF assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assayAgonist activity at human recombinant mu opioid receptor expressed in CHO cells by [35S]GTPgammaS binding assay
Agonist activity at rat MOR expressed in HN9.10 cell membranes by [35S]GTPgammaS binding assayAgonist activity at rat MOR expressed in HN9.10 cell membranes by [35S]GTPgammaS binding assay
Agonist activity at rat mu opioid receptor expressed in HN9.10 cells assessed as stimulation of [35S]GTPgammaS bindingAgonist activity at rat mu opioid receptor expressed in HN9.10 cells assessed as stimulation of [35S]GTPgammaS binding
Stimulation of mouse MOR expressed in CHO cells after 1.5 hrs by [35S]GTPgammaS binding assayStimulation of mouse MOR expressed in CHO cells after 1.5 hrs by [35S]GTPgammaS binding assay
Agonist activity at human mu opioid receptor expressed in CHO-K1 cells assessed as stimulation of [35S]-GTPgammaS binding by scintillation proximity assayAgonist activity at human mu opioid receptor expressed in CHO-K1 cells assessed as stimulation of [35S]-GTPgammaS binding by scintillation proximity assay
Agonist activity at human mu opioid receptor expressed in CHO-K1 cells assessed as stimulation of [35S]-GTPgammaS binding by scintillation proximity assayAgonist activity at human mu opioid receptor expressed in CHO-K1 cells assessed as stimulation of [35S]-GTPgammaS binding by scintillation proximity assay
PubChem BioAssay. Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization. (Class of assay: confirmatory) PubChem BioAssay. Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization. (Class of assay: confirmatory)
Agonist activity at human mu opioid receptor expressed in CHO-dhfr(-) cells assessed as inhibition of forskolin-induced cAMP accumulation by TR-FRET assayAgonist activity at human mu opioid receptor expressed in CHO-dhfr(-) cells assessed as inhibition of forskolin-induced cAMP accumulation by TR-FRET assay
Agonist activity at human MOP receptor expressed in CHO-K1 cells assessed as induction of [35S]GTPgammaS binding after 30 mins by liquid scintillation countingAgonist activity at human MOP receptor expressed in CHO-K1 cells assessed as induction of [35S]GTPgammaS binding after 30 mins by liquid scintillation counting
Agonist activity at rat MOR expressed in CHO cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation countingAgonist activity at rat MOR expressed in CHO cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by liquid scintillation counting
Agonist activity at rat mu opioid receptor assessed as stimulation of [35]SGTPgammaS bindingAgonist activity at rat mu opioid receptor assessed as stimulation of [35]SGTPgammaS binding
Activity at Wistar rat mu opioid receptor assessed as inhibition of low-frequency electrically-stimulated vas deferens contractionActivity at Wistar rat mu opioid receptor assessed as inhibition of low-frequency electrically-stimulated vas deferens contraction
Agonist activity at mu opioid receptor in rat brain membranes assessed as stimulation of [35S]GTPgammaS binding after 60 mins in presence of GDPAgonist activity at mu opioid receptor in rat brain membranes assessed as stimulation of [35S]GTPgammaS binding after 60 mins in presence of GDP
Agonist activity at mu opioid receptor in rat brain membranes assessed as stimulation of [35S]GTPgammaS binding after 60 mins in presence of GDPAgonist activity at mu opioid receptor in rat brain membranes assessed as stimulation of [35S]GTPgammaS binding after 60 mins in presence of GDP
Agonist activity at MOR (unknown origin) expressed in HEK293T assessed as intracellular cAMP accumulation after 15 mins by luciferase based GloSensor assayAgonist activity at MOR (unknown origin) expressed in HEK293T assessed as intracellular cAMP accumulation after 15 mins by luciferase based GloSensor assay
Agonist activity at mu opioid receptor in rat brain membranes assessed as stimulation of [35S]GTPgammaS binding after 60 mins in presence of GDPAgonist activity at mu opioid receptor in rat brain membranes assessed as stimulation of [35S]GTPgammaS binding after 60 mins in presence of GDP
Agonist activity at mu opioid receptor in rat brain membranes assessed as stimulation of [35S]GTPgammaS binding after 60 mins in presence of GDPAgonist activity at mu opioid receptor in rat brain membranes assessed as stimulation of [35S]GTPgammaS binding after 60 mins in presence of GDP
Agonist activity at human mu opioid receptor expressed in CHO cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by scintillation counting methodAgonist activity at human mu opioid receptor expressed in CHO cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by scintillation counting method
PubChem BioAssay. Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization. (Class of assay: confirmatory) PubChem BioAssay. Counterscreen for agonists of heterodimerization of the mu 1 (OPRM1) and delta 1 (OPRD1) opioid receptors: Luminescence-based cell-based high throughput dose response assay to identify agonists of OPRM1 homodimerization. (Class of assay: confirmatory)
Agonist activity at EA-fragment beta-arrestin-tagged human MOR expressed in human U2OS cell membranes assessed as stimulation of [35S]-GTPgammaS binding after 60 mins by beta-galactosidase based scintillation counting methodAgonist activity at EA-fragment beta-arrestin-tagged human MOR expressed in human U2OS cell membranes assessed as stimulation of [35S]-GTPgammaS binding after 60 mins by beta-galactosidase based scintillation counting method
Agonist activity at human MOR expressed in CHOK1 cells assessed as stimulation of cAMP accumulation incubated for 45 mins by HTRF assayAgonist activity at human MOR expressed in CHOK1 cells assessed as stimulation of cAMP accumulation incubated for 45 mins by HTRF assay
Agonist activity at rat mu-opioid receptor expressed in CHO cells after 90 mins by [35S]GTP-gamma-S assayAgonist activity at rat mu-opioid receptor expressed in CHO cells after 90 mins by [35S]GTP-gamma-S assay
Agonist activity at mu opioid receptor (unknown origin) expressed in HEK293-A cells assessed as reduction in forskolin-induced cAMP accumulation incubated for 30 mins by competition PKA binding assayAgonist activity at mu opioid receptor (unknown origin) expressed in HEK293-A cells assessed as reduction in forskolin-induced cAMP accumulation incubated for 30 mins by competition PKA binding assay
Agonist activity at rat mu-opioid receptor expressed in CHO cells after 90 mins by [35S]GTP-gamma-S assayAgonist activity at rat mu-opioid receptor expressed in CHO cells after 90 mins by [35S]GTP-gamma-S assay
HiRange Homogenous Time-Resolved Fluorescence (HTRF) Assay: Briefly, suspensions of cells expressing either the mu, kappa or delta opioid receptors were prepared in buffer containing 0.5 mM isobutyl-methyl xanthine (IBMX). Cells were incubated with varying concentrations of PEG-opioid conjugates and 3 uM forskolin for 30 minutes at room temperature. cAMP was detected following a two-step assay protocol per the manufacturer's instructions and time resolved fluorescence was measured with the following settings: 330 nm excitation; 620 nm and 665 nm emission; 380 nm dichroic mirror. The 665 nm/620 nm ratio is expressed as Delta F % and test compound-related data is expressed as a percentage of average maximum response in wells without forskolin. EC50 values were calculated for each compound from a sigmoidal dose-response plot of concentrations versus maximum response.HiRange Homogenous Time-Resolved Fluorescence (HTRF) Assay: Briefly, suspensions of cells expressing either the mu, kappa or delta opioid receptors were prepared in buffer containing 0.5 mM isobutyl-methyl xanthine (IBMX). Cells were incubated with varying concentrations of PEG-opioid conjugates and 3 uM forskolin for 30 minutes at room temperature. cAMP was detected following a two-step assay protocol per the manufacturer's instructions and time resolved fluorescence was measured with the following settings: 330 nm excitation; 620 nm and 665 nm emission; 380 nm dichroic mirror. The 665 nm/620 nm ratio is expressed as Delta F % and test compound-related data is expressed as a percentage of average maximum response in wells without forskolin. EC50 values were calculated for each compound from a sigmoidal dose-response plot of concentrations versus maximum response.
[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.[35S]GTPγS Functional Assay: [35S]GTPγS functional assays were conducted using freshly thawed μ-receptor membranes prepared from a cell line expressing recombinant μ opioid receptor in a HEK-293, CHO or U-2 OS cell background or purchased from a commercial source (Perkin Elmer, Shelton, Conn.; or DiscovRx, Fremont, Calif.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPγS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 μl/well) was transferred to 96-shallow well polypropylene plates containing 10 μl of 20× concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 200 μl of ice-cold wash buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 μl/well) and plates were counted using a Packard Top-Count for 1 min/well.
Agonist activity at human mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation by fluorescence assayAgonist activity at human mu opioid receptor expressed in HEK293 cells assessed as inhibition of forskolin-stimulated cAMP accumulation by fluorescence assay
Agonist activity at mu opioid receptor (unknown origin) expressed in HEK293-A cells assessed as reduction in forskolin-induced cAMP accumulation incubated for 30 mins by competition PKA binding assayAgonist activity at mu opioid receptor (unknown origin) expressed in HEK293-A cells assessed as reduction in forskolin-induced cAMP accumulation incubated for 30 mins by competition PKA binding assay
Agonist activity at human mu opioid receptor expressed in CHO cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by scintillation counting methodAgonist activity at human mu opioid receptor expressed in CHO cell membranes assessed as stimulation of [35S]GTPgammaS binding incubated for 1 hr by scintillation counting method
Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.Functional Assay: [35S]GTPgammaS functional assays were conducted using freshly thawed u-receptor membranes (Perkin Elmer, Shelton, Conn.). Assay reactions were prepared by sequentially adding the following reagents to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice (final concentrations indicated): membrane protein (0.026 mg/mL), saponin (10 mg/mL), GDP (3 mM) and [35S]GTPgammaS (0.20 nM; Perkin Elmer, Shelton, Conn.). The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of the agonist [D-Ala2, N-methyl-Phe4 Gly-ol5]-enkephalin (DAMGO) prepared in dimethyl sulfoxide (DMSO). Plates were incubated for 30 min at about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by three filtration washes with 2001 of ice-cold binding buffer.
Agonist activity at human MOR expressed in human U2OS cells assessed as increase in beta arrestin2 recruitment incubated for 90 mins by pathhunter beta-arrestin assayAgonist activity at human MOR expressed in human U2OS cells assessed as increase in beta arrestin2 recruitment incubated for 90 mins by pathhunter beta-arrestin assay
Agonist activity at mu opioid receptor (unknown origin) expressed in human HEK293 cells assessed as increase in cAMP accumulation using [3H]-cAMP as substrate measured after 30mins by liquid scintillation counting methodAgonist activity at mu opioid receptor (unknown origin) expressed in human HEK293 cells assessed as increase in cAMP accumulation using [3H]-cAMP as substrate measured after 30mins by liquid scintillation counting method
HiRange Homogenous Time-Resolved Fluorescence (HTRF) Assay: Briefly, suspensions of cells expressing either the mu, kappa or delta opioid receptors were prepared in buffer containing 0.5 mM isobutyl-methyl xanthine (IBMX). Cells were incubated with varying concentrations of PEG-opioid conjugates and 3 uM forskolin for 30 minutes at room temperature. cAMP was detected following a two-step assay protocol per the manufacturer's instructions and time resolved fluorescence was measured with the following settings: 330 nm excitation; 620 nm and 665 nm emission; 380 nm dichroic mirror. The 665 nm/620 nm ratio is expressed as Delta F % and test compound-related data is expressed as a percentage of average maximum response in wells without forskolin. EC50 values were calculated for each compound from a sigmoidal dose-response plot of concentrations versus maximum response.HiRange Homogenous Time-Resolved Fluorescence (HTRF) Assay: Briefly, suspensions of cells expressing either the mu, kappa or delta opioid receptors were prepared in buffer containing 0.5 mM isobutyl-methyl xanthine (IBMX). Cells were incubated with varying concentrations of PEG-opioid conjugates and 3 uM forskolin for 30 minutes at room temperature. cAMP was detected following a two-step assay protocol per the manufacturer's instructions and time resolved fluorescence was measured with the following settings: 330 nm excitation; 620 nm and 665 nm emission; 380 nm dichroic mirror. The 665 nm/620 nm ratio is expressed as Delta F % and test compound-related data is expressed as a percentage of average maximum response in wells without forskolin. EC50 values were calculated for each compound from a sigmoidal dose-response plot of concentrations versus maximum response.
Agonist activity at MOP (unknown origin) expressed in SH-SY5Y cell membranes co-expressing Gbeta1-RGFP protein assessed as induction of G protein activation incubated for 15 mins by BRET assayAgonist activity at MOP (unknown origin) expressed in SH-SY5Y cell membranes co-expressing Gbeta1-RGFP protein assessed as induction of G protein activation incubated for 15 mins by BRET assay
Agonist activity at Rluc-tagged MOR receptor (unknown origin) expressed in human SH-5YSY cells co-expressing RGFP-fused to Gbeta1 assessed as increase in MOR/G beta1 protein interaction incubated for 5 mins in presence of coelenterazine by BRET assayAgonist activity at Rluc-tagged MOR receptor (unknown origin) expressed in human SH-5YSY cells co-expressing RGFP-fused to Gbeta1 assessed as increase in MOR/G beta1 protein interaction incubated for 5 mins in presence of coelenterazine by BRET assay
Agonist activity at EA-fragment beta-arrestin-tagged human MOR expressed in human U2OS cell membranes assessed as stimulation of [35S]-GTPgammaS binding after 60 mins by beta-galactosidase based scintillation counting methodAgonist activity at EA-fragment beta-arrestin-tagged human MOR expressed in human U2OS cell membranes assessed as stimulation of [35S]-GTPgammaS binding after 60 mins by beta-galactosidase based scintillation counting method