5-HT Competition Uptake Assays

Transport of 5-HT was assayed as described previously.³⁵ Briefly, [³H]5-HT (100 nM) and serial dilutions of SERT inhibitors were prepared separately in KRH for synaptosomes or a platelet buffer solution (150 mM NaCl, 50 mM Tris HCl, 25 mM EDTA, pH 7.4, 100 mM pargyline, and 100 mM ascorbic acid) for platelets. The assays were set up on ice in a final volume of 250 μL (150 μL synaptosome or PRP, 50 μL [³H]5-HT, and 50 μL antagonist). The assay was initiated by incubation in a water bath for 10 min at 37 °C followed by immediate filtration over 0.3% polyethylenimine-coated glass fiber filters (Brandel) using a cell harvester (Brandel). Filter-bound radioactivity was quantified by a Beckman LS 6000 liquid scintillation counter. All transport assays were performed in duplicate. Radioactivity was normalized as a percentage of inhibition and plotted to determine the IC₅₀ of the tested antagonist.