Topcount nxt microplate scintillation and luminescence counter

Membranes were prepared from CHO-K1 cells expressing hS1P₁, hS1P₂, hS1P₃, hS1P₄, hS1P₅, rS1P₁, and rS1P₃ based on the methods of Mandala [10] (link) with modifications. Briefly, cells were washed with PBS, suspended in 1 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, and 1× Complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), and disrupted on ice using a dounce homogenizer. The homogenate was centrifuged for 10 min at 1000 g and the supernatant was centrifuged at 100,000 g for 60 min at 4°C. The pellet was suspended in 10 mM Tris-HCl (pH 7.4), 1 mM EDTA and stored at −80°C. ASP4058 and fingolimod-P were dissolved in dimethyl sulfoxide (DMSO) (Wako Pure Chemical Industries, Osaka, Japan) and then diluted with assay buffer (20 mM HEPES [pH 7.5], 100 mM NaCl, 10 mM MgCl₂, 0.1% fatty acid-free bovine serum albumin, and 5 µM GDP) to various concentrations. Membranes (20 µg) were mixed with test-compound solution (final concentration, 1% [v/v] DMSO) and 50 pM [³⁵S]-GTPγS (PerkinElmer, Waltham, MA, USA) in 150 µl of assay buffer. Membranes were incubated for 60 min at room temperature and collected onto GF/B filter plates (PerkinElmer), and then filter-bound radionuclides were measured using a TopCount NXT Microplate Scintillation and Luminescence Counter (PerkinElmer).