Opioid Receptor Binding Assay Protocol

Competitive binding assays were conducted on human opioid receptors stably transfected into CHO cells according to the published procedures [26 (link),27 (link)]. Binding assays were performed using [³H]DAMGO (1 nM), [³H]diprenorphine (0.2 nM), [³H]U69,593 (0.4 nM), or [³H]Nociceptin (0.1 nM) for labeling MOR, DOR, KOR, or NOP receptors, respectively. Non-specific binding was determined using 1–10 µM of the unlabeled counterpart of each radioligand. Assays were performed in 50 mM Tris-HCl buffer (pH 7.4) in a final volume of 1 mL. In NOP receptor experiments, 1 mg/mL bovine serum albumin (BSA) was added to the assay buffer. Cell membranes (15–20 µg) were incubated with various concentrations of test compounds and the appropriate radioligand for 60 min at 25 °C. After incubation, reactions were terminated by rapid filtration through Whatman GF/C glass fiber filters. In the NOP receptor assay, filtration was carried out through 0.5% PEI-soaked Whatman GF/C glass fiber filters. Filters were washed three times with 5 mL of ice-cold 50 mM Tris-HCl buffer (pH 7.4) using a Brandel M24R cell harvester (Gaithersburg, MD, USA). Radioactivity retained on the filters was counted by liquid scintillation counting using a Beckman Coulter LS6500 (Beckman Coulter Inc., Fullerton, CA, USA). All experiments were performed in duplicate and repeated three times with independently prepared samples.