| Assay Method Information | |
| | Detecting Inhibition of cAMP Production |
| Description: | Agonist-promoted G-protein responses were determined by measuring changes in intracellular cAMP using the HTRF cAMP HiRange kit (CisBio, Cat #: 62AM6PEJ) based on time-resolved fluorescence resonance energy transfer (TR-FRET) technology. AssayComplete Cell Plating 11 Reagent was removed and replaced with Ham's F-12 (CellGro, Cat #: 10-080-CM) containing isobutyl-methyl-xanthine (IBMX; 500 μM; Tocris Bioscience, Cat #: 2845), and NKH-477 (1.5 μM; Tocris Bioscience, Cat #: 1603) along with test or control compounds at the desired concentrations. Following a 30-minute incubation at 37 C. and 5% CO2 in a humidified CO2 and temperature-controlled incubator, the components of the cAMP HiRange kit were added as per the manufacturer's instructions. After an hour incubation at room temperature, plates were analyzed by a BMG PheraStar microplate reader. Responses were measured as the ratio of fluorescence emission at 665 nm to fluorescence emission at 620 nm. |
| Affinity data for this assay | |
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