Microscint 0 scintillation cocktail

Membranes (20 μg per well diluted in 50 mM Tris-HCl, pH 7.4) were incubated with [³H] PK 11195 (Perkin-Elmer, Beaconsfield, UK) at seven concentrations (0.1–25 nM), 3 μM unlabelled PK 11195 (Sigma-Aldrich) to determine non-specific binding, or a vehicle control (0.1% DMSO) (v/v) to determine total binding. The reaction was incubated for 90 min at 4 °C to achieve equilibrium, terminated by rapid filtration through a glass-fibre filter (GF/C; Millipore, Carrigtwonhill, Ireland) and washed with 50 mM Tris-HCl (pH 7.4) at 4 °C. The filters were allowed to dry overnight, covered with Microscint-0 scintillation cocktail (Perkin-Elmer) and radioactivity measured with a Microbeta² 2450 Microplate Counter (Perkin-Elmer). Saturation binding was analysed with Graphpad Prism 6.0 (Graphpad, La Jolla, CA) using a non-linear regression curve fit to determine the K_d. Data are expressed as mean ± SEM from at least three independent experiments.

Membranes (20 μg per well diluted in 50 mM Tris-HCl, pH 7.4) were incubated with ∼K_d concentration of [³H]PK 11195 (10 nM) and test compounds (0.3 nM–10 μM) in a final volume of 200 μL for 90 min at 4 °C. To determine total binding, [³H]PK 11195 and membranes were incubated with vehicle alone (0.1% DMSO (v/v) in 50 mM Tris-HCl, pH 7.4). Non-specific binding was determined in the presence of a saturating concentration of cold PK 11195 (3 μM), and amounted to 5–15% of total binding. After incubation, assays were terminated by rapid filtration through a 96-well glass-fibre filter plate (Millipore) and washed 8 times with ice-cold 50 mM Tris-HCl (pH 7.4) using a Brandel 96-sample vacuum harvester. The filter was covered with MicroScint 0 scintillation cocktail (Perkin Elmer) and radioactivity counted using a Microbeta² 2450 Microplate Counter (Perkin Elmer). Data were corrected for non-specific binding and expressed as a percentage of vehicle control. Data were analysed using Graphpad Prism 6.0 (Graphpad) and a four-parameter non-linear regression curve fit to calculate the K_i values. Data are expressed as mean ± SEM from at least three independent experiments.