CB1 and CB2 Receptor β-Arrestin Recruitment Assay

Assays were carried out using the PathHunter hCB₁_bgal, hCB₂_bgal, or mCB₂_bgal CHOK1 β-arrestin recruitment assay kit (DiscoveRx, Fremont, CA, USA) as previously described [28 (link)]. A total of 5000 cells per well were seeded in 384-well plates (Perkin Elmer, MA, USA) containing 20 μL cell culture medium and incubated for 16–18 h at 37 °C and 5% CO₂. Cells were stimulated with 10 μM of each agonist or 11 increasing concentrations and incubated for 90 min at 37 °C and 5% CO₂. In the inverse agonist assays, cells were exposed to 10 μM of RO6851228 or 11 increasing concentrations and incubated for 30 min at 37 °C and 5% CO₂, followed by the addition of the EC₈₀ concentration of CP55940 (25 nM for CHOK1hCB₁_bgal and 46 nM for CHOK1hCB₂_bgal). The cells were incubated for 90 min at 37 °C and 5% CO₂. Compounds in DMSO stock solutions were added using a HP D300 Digital Dispenser (Tecan, Mannedorf, Switzerland). The final concentration of organic solvent per assay point was ≤ 0.1%. The PathHunter Detection mixture was used, according to the manufacturer’s protocol, to determine the β-galactosidase enzyme activity. Detection mixture, 12 μL per well, was added and the plate was incubated for 1 h in the dark at room temperature. Chemiluminescence, indicated as relative light unit, was measured on an EnVision multilabel plate reader (Perkin Elmer, MA, USA).