Microbeta 2450 microplate counter

The cellular background HEK293 containing 400 μL of h-SERT (Code RBHSTM400UA, Perkin-Elmer, Santiago, Chile) was diluted in 12 Eppendorf tubes, containing a storage buffer solution of Tris-HCl 50 mM (pH 7.4), EDTA 0.5 mM, MgCl₂ 10 mM and 10% sucrose, obtaining a final volume between 260 and 340 μL, which was finally stored at −80 °C [46 (link)].
Each Eppendorf tube was incubated with 50 mM Tris HCl buffer (pH 7.4), 120 mM NaCl, 5 mM KCl and the drugs under study using increasing concentrations, in presence of 2 nM of [³H]-paroxetine (specific activity 23.1 Ci/mmol, Code NET86925UC, Perkin-Elmer, Santiago, Chile) with a final volume of 250 μL [46 (link)]. Non-specific binding was determined using 25 mM fluoxetine. After 30 min at 27 °C, the incubation was stopped by rapid filtration on a Whatman GF/C filter preabsorbed in 0.5% polyethylenimine (PEI), washed with cold working buffer solution, 3 × 3mL, filtered, and scintillation liquid was added. The radioactivity was measured by liquid scintillation spectrometry (MicroBeta 2450 microplate counter, PerkinElmer, Santiago, Chile). The data were represented in a bar graph using 25 μM concentration for both, the compound under study and fluoxetine as the non-specific ligand. Each bar represents the mean ± S.E.M obtained in the experiments carried out in triplicates.

To determine the binding of all compounds at SERT, competitive binding assays were performed according to previously reported procedures with some modifications [36 (link)]. Briefly, assays were carried out in a total volume of 0.5 mL containing 9 µg protein of membrane from a clonal cell line HEK-293 that overexpresses SERT, 50 mM Tris buffer, pH 7.4, 120 mM NaCl, 5 mM KCl, 2 nM [³H]-paroxetine (specific activity 20.8 Ci/mmol, PerkinElmer), and the compounds to be tested at different concentrations (10⁻⁹–10⁻⁴ M). After 1 h at 27 °C, incubations were stopped by rapid filtration through Whatman GF/C filters presoaked in 0.5% polyethyleneimine, which were washed five times with 3 mL of ice-cold buffer, dried, and put in Eppendorf tubes with scintillation liquid. Radioactivity was counted by a liquid scintillation counter (MicroBeta 2450 microplate counter, PerkinElmer). Control curve was performed with fluoxetine in the same experimental conditions. Non-specific binding was determined with 10 µM fluoxetine.

The cellular background CHO-K1 with 400 μL of h-DAT (Code RBHDATM400UA, Perkin-Elmer, Santiago, Chile) was diluted in 12 Eppendorf tubes, with a storage buffer solution of Tris-HCl 50 mM (pH 7.4), EDTA 0.5 mM, MgCl₂ 10 mM and 10% sucrose, obtaining a final volume of 260–340 μL stored at −80 °C [46 (link)]. Each Eppendorf tube was incubated in 50 mM Tris-HCl buffer solution (pH 7.4), 100 mM NaCl, the drugs under study were using increasing concentrations, with 1 nM of [³H]-WIN 35,428 (specific activity 82.6 Ci/mmol, Code NET1033025UC, Perkin-Elmer, Santiago, Chile) with a final volume of 250 μL. Non-specific binding was determined using 10 mM methylphenidate.
After 120 min at 4 °C, the incubation was stopped by rapid filtration on a Whatman GF/C filter preabsorbed in 0.5% polyethylenimine (PEI), washed with cold working buffer solution, 3 x 3mL, filtered, and scintillation liquid was added. The radioactivity was measured by liquid scintillation spectrometry (MicroBeta 2450 microplate counter, PerkinElmer, Santiago, Chile). The data were plotted by non-linear regression variable inhibitor-response dose (Prism 5.01, GraphPad, San Diego, CA, USA) to estimate the IC₅₀ and K_i values for the tested compounds using the Cheng-Prusoff equation.