Cryopreserved HEK-ACΔ3/6 cells overexpressing human AC1, AC2, AC5 or AC8 were transferred to a 15-mL Falcon tube and gently resuspended in prewarmed Opti-MEM. The cells were centrifuged for 5 minutes at 150 x g, supernatant was discarded, and the cell pellet was resuspended in 10 mL for a second centrifugation step. Then, the cells were counted, plated in a white opaque 384-well plate and incubated for 1-hour in a 37°C incubator supplemented with 5% CO2. The hit compounds' working solutions were prepared in prewarmed Opti-MEM and successive serial dilutions were prepared using a Precision 2000 automated pipetting system. Dose response curves were generated for each hit compound using a three-fold serial dilution starting at 30 μM concentration (at least 6-points per curve). Compounds were added to the assay plates and incubated for 30 minutes at room temperature. AC activity was selectively stimulated on each AC isoform with 3 μM A23187 (AC1 and AC8), 100 nM PMA (AC2), or a low concentration (1 μM)