MTT Assay for Melanoma Cell Viability

A cell viability was determined using MTT assay to estimate the probable cytotoxic effect of hdTIPs and positive controls (kojic acid and arbutin) on melanoma cells. The number of viable cells was determined by the ability of mitochondria to convert MTT to formazan dye. The quantity of formazan formed is proportional to the number of viable cells present and can be measured spectrophotometrically. The method for MTT assay in this study was modified from Zaidi et al. [74 (link)]. Briefly, when cells density from Section 4.2 reached 70% of culture flask, the remaining adherent cells were trypsinized for 5 min, counted by a hemocytometer and seeded into 96-well plates at 10 × 10⁴ cells/mL, then incubated overnight. The cells were then treated with each hdTIP candidates (TIP1, TIP2, KNN1, KNN2, KNN3, RF1, RF2, and RF3) at various concentrations of 25, 50, 100, 200 μM, along with the two positive controls, kojic acid (KA) and arbutin at various concentrations (0, 125, 150, 500, and 1,000 μg/mL). After 24 h incubation, 1 mg/mL of MTT (Invitrogen, Eugene, USA) solution was replaced prior to incubation at 37 °C for 3 h. The formazan precipitates were dissolved by 100 μL of dimethyl sulfoxide (DMSO) and the concentrations were measured at 570 nm in a microplate reader (Synergy H1, BioTek, Santa Clara USA). Cell viability was calculated using the following formula: cell viability (%) = (A_sample/A_control) × 100, where A_sample and A_control are the absorbances from the mixture with or without the addition of test sample, respectively.