CB1 and CB2 Receptor Binding Assay

Competition receptor binding was performed as reported earlier [30 (link)]. Briefly, each reaction mixture contained either 100 μg of CHO-hCB1-Rx or 50 μg of CHO-hCB2 membrane homogenates, 0.2 nM [³H]-CP55,940, 5 mM MgCl₂, and increasing concentrations of the non-radioactive competing ligands in a 50 mM Tris-HCl buffer (pH 7.4) with 0.1% bovine serum albumin. The total volume of the incubation mixture was 1 ml. All reactions were mixed and allowed to reach equilibrium binding by incubation at room temperature for 90 min. Non-specific binding was defined as the amount of radioligand binding remaining in the presence of a 1 μM concentration of the non-radioactive, high affinity, CB1/CB2 agonist WIN-55,212–2. Binding was terminated by rapid vacuum filtration through glass fiber filters (Brandel, Gaithersburg, MD), followed by four 5 ml washes of ice-cold 50 mM Tris-HCl (pH 7.4) buffer containing 0.1% bovine serum albumin. Four ml of scintiverse scintillation fluid (Fisher Scientific, Waltham, MA) was added to the filters and the amount of radioactivity was quantified 24 hr later utilizing liquid scintillation spectrophotometry.