4.3. 5-HT1A Receptor Binding Assays

10 μg of membranes from CHO-K1 cells expressing the human 5-HT1A receptor were suspended in 50 mM Tris-HCl, 10 mM MgSO₄, 0.5 mM EDTA, and 0.1% (w/v) ascorbic acid, pH 7.4 and test ligands (91 nM for single point radioligand displacement assay and 1 nM to 1 μM for full competition assay) were incubated with 0.25 nM [³H]8-hydroxy-DPAT (PerkinElmer, Boston, MA, USA) for 60 min at room temperature. The nonspecific binding was determined using 10 μM metergoline (Sigma, St. Louis, MO, USA). After incubation, the bound ligands were filtered using an M-24 Brandel filtration system (Brandel, Gaithersburg, MD, USA), collected on glass fiber papers (Whatman grade 934-AH, GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), and the radioactivity was quantitated using a MicroBeta2 Microplate scintillation counter 2450 (PerkinElmer, Boston, MA, USA).