| Assay Method Information | |
| | cAMP Inhibition Assay |
| Description: | HEK293T cells were co-transfected with plasmids encoding the cAMP biosensor GloSensor-22F (Promega) and the different human KOR variants as described [Allen et al., Proc. Natl. Acad. Sci. U.S.A., 108:18488-18493]. After an 18-h incubation at 37 °C, the cells were seeded (at 20,000 cells/20 μl/well) into white, clear bottom, 384-well tissue culture plates precoated with poly-L-lysine (25 mg/liter; Sigma). Cells were plated in DMEM containing 1% dialyzed FBS. After a 24-h recovery, the medium was replaced with 20 μl of Drug Buffer (1× Hanks' balanced salt solution, 20 mM HEPES, pH 7.4), and the cells were treated with 10 μl of 3× test or reference drug prepared in Drug Buffer. After 20 min, cAMP production was stimulated and detected by treatment with 10 μl of 1.2 μM (4×) isoproterenol in 8% (4×) GloSensor reagent. Luminescence per well per second was read on a Wallac Micro-Beta TriLux plate scintillation counter. |
| Affinity data for this assay | |
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