Reuptake assay

Reuptake assay was conducted using the HEK-293 cells stably expressing DAT, NET, and SERT. Cells were cultured in 24-well plates and washed with an uptake buffer comprising 5 mM Tris base, 7.5 mM HEPES, 120 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl₂, 1.2 mM MgSO₄, 1 mM ascorbic acid, and 5 mM glucose (pH 7.1). Thereafter, the cells were treated with the synthesized compounds at 37°C for 20 min, followed by incubation with [³H]-DA or [³H]-5-HT (final concentration, 20 nM) for 5 min. The cells were then washed thrice with ice cold uptake buffer and incubated overnight with phosphate-buffered saline containing 1% sodium dodecyl sulfate. Post incubation, samples were mixed with a scintillation cocktail and radioactivity was measured using the Wallac 1450 MicroBeta^® TriLux liquid scintillation counter (PerkinElmer). GBR12909 and venlafaxine were used as the reference compounds for the DA and NE/5-HT reuptake assay, respectively.

To determine the IC₅₀ of the synthesized compounds, the concentration of the compounds was gradually increased and the percentage of inhibition was assessed for each concentration. GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA) was used to construct the dose-response curve and determine the IC₅₀.