| Assay Method Information | |
| | Homogeneous Time-Resolved Fluorescence (HTRF) Assay |
| Description: | HTRF cAMP assays were performed using commercially available assay kits according to the manufacturer's instructions (cAMP Dynamic 2 Assay Kit; #62AM4PEJ, Cisbio Bioassays, Bedford, MA). An aliquot of CHO-K1 cells stably expressing recombinant human GPR52 was thawed and resuspended in cell buffer (1×PBS (w/o Ca2+/Mg2+)) at a density of 4×105 cells per mL. Test compounds were solubilized in DMSO to 10 mM stock solutions and serially diluted in DMSO using 6-fold dilutions to generate 8-point dose response curves. These serially diluted samples were then diluted 1:50 in compound dilution buffer (1×PBS (w/o Ca2+/Mg2+) containing 0.5 mM IBMX, 0.1% BSA) to achieve a 4× stock. The diluted compounds were transferred (5 μL per well) in duplicate to the 384-well assay plate (Optiplate #6007290, PerkinElmer, Waltham, MA). Both a positive (reference compound) and negative (non-stimulated vehicle) control was included in each assay run in column 23. The cell suspension was subsequently dispensed into the 384-well assay plates at 15 μL per well (6000 cells) such that the compound was diluted to 1×. Column 24 on the plates did not receive cells and was reserved for a cAMP standard curve. After a one-hour incubation at room temperature, 10 μL of cAMP D2 reagent followed by 10 μL of cryptate reagent (provided in the Cisbio kit) was added to each well. Plates were then incubated at room temperature for one hour prior to reading. Time-resolved fluorescence measurements were collected on an EnVision® HTRF plate reader (PerkinElmer, Waltham, MA). |
| Affinity data for this assay | |
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