2.3. Radioligand Binding Experiments

In 35 mm dishes, the SH-EP1 cells were grown to confluence, collected by scraping in 50 mM HEPES buffer solution containing 1 mM MgCl₂, 2.5 mM CaCl₂, 0.1% (w/v) bovine serum albumin, 0.025% (w/v) bacitracin, and 0.025% (w/v) sodium azide (pH 7.4), and centrifuged at 1200 r.p.m. for 15 min at 4 °C. Subsequently, the supernatant was removed and cells were frozen at −80 °C until the day of the experiment. For binding assays, using a Polytron tissue homogenizer at setting 4 for 20 s, the cells were resuspended in 50 mM Tris-HCl buffer containing 120 mM NaCl, 5 mM EDTA, 1.5 mM MgCl₂, and 5 mM KCl (pH 7.4). In a total of 250 µL volume, 150 µL cell suspension, 50 µL radioligand [¹²⁵I]-α-bungarotoxin (2200 Ci/mmol; Perkin-Elmer, Inc. Waltham, MA, USA) and 50 µL test compound, were added to 96-well microtitre plates. The α-bungarotoxin (3 µM) was used to determine non-specific binding. Subsequent to 45 min incubation at room temperature, the plates were filtered through Packard Unifilter-96, GF/C plates and washed twice with 500 µL ice-cold 10 mM Tris-HCl buffer containing 150 mM NaCl (pH 7.4). The radioactivity bound to filters was counted in 50 µL of scintillation solution (MicroScint 40, Perkin-Elmer, Inc. Waltham, MA, USA) in Packard TopCount scintillation counter. Assays were performed in triplicate.