Tricarb 2900tr scintillation counter

Membranes from cells stably expressing KOR, MOR, or DOR constructs (PathHunter U2OS hOPRK1, CHO-K1 rOPRM1, and CHO-K1 OPRD1 β-arrestin-2 cell line, DiscoverX) were used. Cells were scraped from tissue culture plates, homogenized with a tissue tearor homogenizer in membrane buffer (10 mM Tris, 100 mM NaCl, and 1 mM EDTA; pH 7.4), and centrifuged at 20000 g for 30 minutes at 4°C and frozen at -80°C until use. Prior to use, the pellets were resuspended in binding buffer (50 mM Tris-HCl, pH 7.4), homogenized with a dounce homogenizer, and 50 μg incubated with 1.0 nM of the appropriate tritiated ligand ([³ H]U69,593, [³ H]DAMGO, or [³ H]DPDPE for kappa, mu, or delta binding, respectively) and the appropriate concentration of compound for 60 minutes at 30°C. Nonspecific binding of radioligand to KOR, MOR, or DOR was determined in the presence of 10 µM norbinaltorphimine, naloxone, or naltrindole, respectively. Membranes with bound tritiated ligand were collected on Whatman GF/B filter paper (Brandel) utilizing a Brandel harvester. Bound tritiated ligand was quantified using a TriCarb-2900TR scintillation counter (Packard) following addition of 4 mL ReadySafe scintillation fluid (Beckman Coulter).

Membranes from U2OS cells (PathHunter U2OS hOPRK1) stably expressing human kappa opioid receptors were used. Cells were scraped from tissue culture plates, homogenized with a tissue tearor homogenizer in membrane buffer (10 mM Tris, 100 mM NaCl, and 1 mM EDTA; pH 7.4), and centrifuged at 20000 g for 30 minutes at 4°C and frozen at -80°C until use. Prior to use, the pellets were resuspended in assay buffer (50 mM Tris, 100 mM NaCl, 5 mM MgCl₂, and 1 mM EDTA; pH 7.4) and homogenized with a dounce homogenizer and 50 μg incubated with 0.1 nM [³⁵ S]GTPγS, 10 nM GDP, and the appropriate concentration of agonist for 20 minutes at 30°C. Membranes with bound [³⁵ S]GTPγS were collected on Whatman GF/B filter paper (Brandel) utilizing a Brandel harvester. Bound [³⁵ S]GTPγS was quantified using a TriCarb-2900TR scintillation counter (Packard) following addition of 4 mL ReadySafe scintillation fluid (Beckman Coulter).