White area microplate

Stimulation buffer containing 1× Hank’s Balanced Salt Solution (HBSS), 5 mM HEPES, 0.1% BSA stabilizer, and 0.5 mM final IBMX was prepared and titrated to pH 7.4 at room temperature. Serial dilutions of the test compounds (5 μL) and 300 nM forskolin (5 μL), both prepared at 4× the desired final concentration in 2% DMSO/ stimulation buffer, were added to a 96-well white ½ area microplate (PerkinElmer). A cAMP standard curve was prepared at 4× the desired final concentration in stimulation buffer, and 5 μL was added to the assay plate. Stable PPLS-HA-GPR88 CHO cells were lifted with versene and spun at 270g for 10 min. The cell pellet was resuspended in stimulation buffer and 4000 cells (10 μL) were added to each well except wells containing the cAMP standard curve. After incubating for 30 min at room temperature, Eu-cAMP tracer and uLIGHT-anti-cAMP working solutions were added per the manufacturer’s instructions. After incubation at room temperature for 1 h, the TR-FRET signal (ex 337 nm) was read on a CLARIOstar multimode plate reader (BMG Biotech, Cary, NC).

All cAMP assays were performed using our previously published methods.^{26 (link)} Stimulation buffer containing 1X Hank’s Balanced Salt Solution (HBSS), 5 mM HEPES, 0.1% BSA stabilizer, and 0.5 mM final IBMX was prepared and titrated to pH 7.4 at room temperature. Serial dilutions of the test compounds (5 μL) and 300 nM forskolin (5 μL), both prepared at 4x the desired final concentration in 2% DMSO/stimulation buffer, were added to a 96-well white ½ area microplate (PerkinElmer). A cAMP standard curve was prepared at 4x the desired final concentration in stimulation buffer and 5 μL was added to the assay plate. Stable PPLS-HA-GPR88 CHO cells were lifted with versene and spun at 270g for 10 minutes. The cell pellet was resuspended in stimulation buffer and 4,000 cells (10 μL) were added to each well except wells containing the cAMP standard curve. After incubating for 30 min at room temperature, Eu-cAMP tracer and uLIGHT-anti-cAMP working solutions were added per the manufacturer’s instructions. After incubation at room temperature for 1 hour, the TR-FRET signal (ex 337 nm) was read on a CLARIOstar multimode plate reader (BMG Biotech, Cary, NC).