Rna binding ysi spa beads

CHO-K1 cells stably expressing human 5-HT_2B receptor (CHO-K1-5-HT_2B) cells were seeded into 96-well tissue culture plates at a density of 20000 cells/well. After 24 h, growth medium was replaced by serum-free medium containing 10 μCi/mL [³H]myo-inositol (20.3 Ci/mmol, Perkin-Elmer) for 24 h. After a labelling period of 24 h, cells were washed for 10 min at 37 °C with Hanks’ balanced salt solution (HBSS) supplemented with 20 mM HEPES, 20 mM LiCl and 2% fatty acid free bovine serum albumin (BSA) (assay buffer) and incubated in assay buffer in the absence (vehicle) or in the presence of the indicated concentrations of the compounds for 20 min at 37 °C. After the incubation time, assay buffer was discarded and 200 μL of 100 mM formic acid was added to the cells for 30 min at 4 °C and 20 μL of the lysate were transferred to a flexiplate plate (Perkin-Elmer, Waltham, MA, USA) together with 80 μL of a solution of RNA Binding YSi SPA Beads (Perkin-Elmer, Waltham, MA, USA) to measure accumulation of [³H]IPs (IP1,IP2, and IP3, collectively referred to as IPs). Radioactivity was quantified in a liquid scintillation counter WALLAC Microbeta TriLux 1450-023.