Cell membrane preparation and radioligand-binding assay

Membranes were prepared from HEK293T cells transiently expressing hSERT growing on 15-cm tissue culture plates. At 40–48 h post transfection, cells were suspended in PBS and centrifuged at low speed (700g) for 5 min. The cell pellet was resuspended in cold H₂O and frozen at −20 °C for 1 h. The suspension was then thawed on ice and subjected to 10–15 passages through a 21-Ga needle to disrupt cells. The homogenate was centrifuged at 18,000g at 4 °C for 30 min, the supernatant fraction was aspirated and the pellet was resuspended in PBSCM. Protein concentration was determined by the BCA method using Pierce bicinchoninic acid (BCA) Protein Assay (ThermoFischer). Membranes were used directly for binding assays or stored at −80 °C until use. For saturation binding studies, increasing concentrations of [³H]imipramine or [³H]vortioxetine were incubated with 5 or 15 μg total membrane protein per sample in a total volume of 150 μl PBSCM. Binding proceeded for 2 h on ice with gentle rocking. Subsequently, membranes were transferred to 96-well glass fibre filter plates preincubated with 0.1% polyethyleneimine (30 μl per well) using a Packard Bell cell harvester and washed four times with water. Non-specific binding was determined in parallel with membranes from non-transfected HEK293T cells. Filter plates were dried and soaked in scintillation fluid (MicroScint-0), followed by counting in a Packard Topcounter. Saturation binding assays were carried out in triplicate and repeated at least three times.