Meltilex scintillant

The receptor binding assay was performed using CHO-K1 cells expressing various TRPV1 mutants (wild-type, Y511A, F587A, F591A and L670A). Whole cell fractions were incubated with 1,000 or 10,000 pM [³H]-RTX (Perkin Elmer, Waltham, MA, USA). Non-specific binding was evaluated in the presence of 1 μM RTX. After 1 h, assays were harvested onto GF/C filtermats using a Filtermate harvester (Perkin Elmer). Then, MeltiLex scintillant (Perkin Elmer) was melted onto dried filtermats and the residual bound radioligand was measured by scintillation counting in a TriLux microbeta counter (Perkin Elmer).

Human MOR, DOR, or KOR recombinant CHO cell membranes were incubated for 2 h at 25 °C in 0.25 mL of 50 mM Tris–HCl buffer containing 5 mM MgCl₂, 1 mM EGTA, 100 mM NaCl, pH 7.4 with various concentrations of the tested compound, 30 μM guanosine 5-diphosphate (GDP) and 0.1 nM [³⁵S]GTPγS (PerkinElmer, Inc.). The incubation was terminated by collecting membranes on Filtermat B filter (PerkinElmer, Inc.) using a FilterMate™ harvester (Perkin Elmer, Inc.). The filters were then washed three times with 50 mM Tris–HCl buffer, pH 7.4. Then, MeltiLex scintillant (PerkinElmer, Inc.) was melted onto the dried filters. Radioactivity was determined by a MicroBeta scintillation counter (PerkinElmer, Inc.). EC₅₀, E_max, and 95% CI values were calculated by Prism software (version 5.0). Nonspecific binding was measured in the presence of 10 μM unlabeled GTPγS (PerkinElmer, Inc.). DPDPE and ICI-174,864 (PerkinElmer, Inc.) were used as the standard DOR full agonist and full inverse agonist, respectively.

Human MOR, DOR, or KOR recombinant CHO cell membranes were incubated for 2 h at 25 °C in 0.25 mL of the buffer containing with various concentrations of the tested compound, 2 nM [³H] DAMGO, [³H] DPDPE, or [³H] U69,593 (PerkinElmer, Inc., MA, USA), respectively. The incubation was terminated by collecting membranes on Filtermat B filter (PerkinElmer, Inc.) using a FilterMate™ harvester (PerkinElmer, Inc.). The filters were then washed three times with 50 mM Tris–HCl buffer, pH 7.4. Then, MeltiLex scintillant (PerkinElmer, Inc.) was melted onto the dried filters. Radioactivity was determined by a MicroBeta scintillation counter (PerkinElmer, Inc.). Nonspecific binding was measured in the presence of 10 μM unlabeled DAMGO, DPDPE, or U-69,593 (PerkinElmer, Inc.). K_i and 95% CI values were calculated by Prism software (version 5.0).