Radioligand Binding Assay for CB1 and CB2

Increasing concentrations of the GBD compounds were incubated with 0.2 nM of the nonselective CB₁/CB₂ agonist [³H]CP-55,940 in a final volume of 1 mL of the binding buffer (50 mM Tris, 0.05% bovine serum albumin, 5 mM of MgCl₂, pH 7.4) as described previously.^{66 (link)} Each binding assay contained 100 or 50 μg of membrane protein prepared from CHO-hCB1 or CHO-hCB₂ cells, respectively. Reactions were incubated for 90 min at rt. Nonspecific binding was defined as binding observed in the presence of 10 μM of the nonselective CB₁/CB₂ ligand WIN-55,212-2. Reactions were terminated by rapid vacuum filtration through Whatman GF/B glass fiber filters, followed by three washes with the ice-cold binding buffer. Filters were then immediately placed into scintillation vials with 4 mL of the scintiverse™ BD cocktail scintillation fluid (Fisher Scientific, Fair Lawn, NJ). Samples were incubated overnight in the scintillation fluid, and vortexed and bound reactivity was determined by employing a liquid scintillation spectrophotometer (Tri Carb 2100 TR Liquid Scintillation Analyzer, Packard Instrument Company, Meriden, CT).