Cell Viability Evaluation via MTT Assay

Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously [18 (link)]. First, the cells were seeded in 96-well plates (20000 cells/well) and cultured for 24 h in medium. After overnight incubation, the medium was changed, and the investigated sample at a concentration varying from 0.03 to 0.25 mg/mL was added for 24 h. The negative control was represented by cells cultivated in a medium without the investigated compounds. Following incubation, the medium was changed, and the MTT solution was added to each well to a final concentration of 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. The absorbance of the samples was recorded at 570 nm using a plate reader Mithras 940 (Berthold). The data were corrected for the background, and the percentage of viable cells was obtained using the equation:
The half-maximal inhibitory concentration (IC₅₀) was determined by fitting the data with a sigmoidal logistical equation using the software Origin 8.1 (Microcal Inc., Los Angeles, CA, USA).