Cell Viability Evaluation using MTT Assay

Cell viability was evaluated using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) as previously described [14 (link)]. Briefly, BJ and B16 cells were seeded in 96-well plates at densities of 10,000 cells/well and 7000 cells/well and further cultured in the medium for 24 h. The next day, the investigated compounds were added at a concentration of 1 to 25 μM for 24 and 48 h. The cells cultivated in medium without the studied samples served as negative controls. After incubation for the desired times, the medium was replaced with MTT solution (1 mg/mL) and incubated for an additional 4 h at 37 °C. Finally, the medium was collected, and DMSO was used to dissolve the insoluble formazan product. A Mithras 940 (Berthold, Bad Wildbad, Germany) plate reader was used to measure the absorbance of the samples at 570 nm. The data were corrected for background, and the percentage of viable cells was obtained using the following equation:
Half-maximal inhibitory concentration (IC₅₀) was determined by fitting the data with a sigmoidal logistic function using Origin 8.1 (Microcal Inc., Northampton, MA, USA) software.