*4.5. Intracellular cAMP Assay with cADDis®*

The assay was performed as described previously [53]. In brief, cells were seeded at 7.0 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/well on black-walled, clear flat-bottom 96-well plates with recombinant BacMam virus expressing the cADDis sensor and 0.6 µM sodium butyrate, and incubated for 24 h at 5% CO<sup>2</sup> at 37 ◦C. The medium was replaced with 100 µL Krebs solution or pretreatment reagents. The 96-well plates were incubated at 28 ◦C for 30 min in the dark. Cell fluorescence was measured from the bottom of the plate using excitation/emission wavelengths of 485 and 525 nm, respectively, on FlexStation 3 (Molecular Devices, LLC., San Jose, CA, USA). Cells were stimulated with 50 µM forskolin to increase the cAMP levels. After 20 min, when the signal plateaued, cells were stimulated with the indicated drugs, and changes in fluorescence from each well were measured every 26 s for 40 min. Increase in fluorescence intensity reflects the decrease in cAMP, through the activation of Gi-coupled receptor. The data were transformed to changes in fluorescence over the initial fluorescence (∆F/F0).
