2.4. MTT Assay for Assessment of Cell Viability

Cell viability, which would reveal the cytotoxic property of the extract, was evaluated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay with slight modifications [17]. Cells were seeded in 96-well plates at 5 × 10³ cells/well and incubated for 24 h at 37°C. The cells were treated with the extract at increasing concentrations within 0–1000 µg/mL for 24 h, at 37°C. The extract was quantitatively dissolved in minimum quantity of DMSO and diluted in the culture medium to prepare the stock solution, which was then made up in the culture medium so as to have the final extract at DMSO concentration of 0.1%. This concentration of DMSO is known not to affect the cell viability [18]. DMSO at this concentration was also used as the solvent control. Experiments with each extract concentration were conducted in triplicates on the same batch of cells. After 24 h incubation, 20 mL of MTT (Sigma-Aldrich, St. Louis, MO, USA) solution (5 mg/mL in PBS) was added to each well and incubated for 3 h at 37°C. The medium was then removed, and 100 mL of DMSO was added to each well to dissolve the purple formazan product. The absorbance was measured at 570 nm (measurement) and 630 nm (reference) using a 96-well plate reader (Bio-Rad, Hercules, CA, USA). The percentage inhibition was calculated from these data using the following formula, and IC₅₀, defined as concentration of the test substance at which cell viability is decreased to 50%, was calculated.