MTT Assay for API Cytotoxicity

The effect of API (purchased from Sigma-Aldrich) on SK-MEL-24 human melanoma cells viability was evaluated by using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The method was performed as previously described [37 (link)]. In brief, a number of 1 × 10⁴ cells/well were seeded onto 96-well culture plates and allowed to adhere overnight. The next day, the cells were stimulated with different concentrations of API (0.3, 1, 3, 10, 30, and 60 μM) and further incubated for 72 h. After 72 h of incubation, a volume of 10 μL of 5 mg/mL MTT solution from the MTT kit (Sigma-Aldrich) was added in each well and incubated for an additional 3 h. The formed formazan crystals were dissolved by adding 100 μL/well of lysis solution provided in the MTT kit. The cells treated with the solvent dimethyl sulfoxide (DMSO, Sigma-Aldrich) were used as the control group. The absorbance was spectrophotometrically analyzed at 570 nm with a microplate reader (BioRad, xMark Microplate Spectrophotometer).