MTT assay and estimation of half-maximal inhibitory concentration (IC50) values

Transfected and non-transfected H1299 cells were seeded at a density of 3.5×10³ cells/well in 96-well plates and incubated overnight at 37°C. CDDP was added at numerous concentrations: 0.01, 0.05, 0.25, 1.25, 10, 20 and 30 µmol/l. CDDP was administered for 48 h at 37°C. The MTT (Sigma-Aldrich; Merck KGaA) reagent (20 µl of 5 mg/ml) was added and incubated for 4 h at 37°C. The reaction was stopped by the addition of 150 µl dimethyl sulfoxide. The optical density at a wavelength of 570 nm was determined on a microplate reader and the untreated cells were used as the control for 100% viability. The IC₅₀ values were calculated from the dose-response data and was the concentration required to inhibit cell growth by 50% compared with the untreated control. Values were estimated using dose-concentration curves showing the percent of cell count.