Cellular Viability Evaluation via MTT Assay

To test cellular viability, we used the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay as previously described [51 (link)]. Briefly, L929 and B16 cells were seeded in 96-well plates at a density of 5000 cells/well and grown in the culture medium for 24 h. The next day, the cells were treated with the investigated compounds, added in concentrations of 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, and 25 μg/mL for 24 and 48 h, respectively. The cells cultivated without the tested compounds served as negative controls. After the incubation period, the culture media was removed, and the MTT solution was added at a final concentration of 0.5 mg/mL. This assay is based on transforming the tetrazolium salt into formazan crystals in the metabolically active cells. After 4 h of incubation at 37 °C, the formazan crystals were dissolved using DMSO. The absorbance of the samples was recorded at λ = 570 nm using a plate reader (Mithras 940, Berthold, Bad Wildbad, Germany). The data were corrected for background absorbance, and the viability was calculated using the following formula:
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.