MTT cell viability assay

Human colon cancer cell lines (DLD-1, HCT-15, and HCT-116) were seeded in 96-well plates at a density of 3000 cells per well. The next day, cells were treated as indicated and incubated at 37°C for a period of 24–72 h. Then, 25 μl of 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, USA) was added to each sample in a volume of 100 μl and incubated for 4 h. After that, 150 μl of N, N-dimethylformamide (Sigma, USA) solubilization solution was added to each well to dissolve the formazan. The absorbance was read at a wavelength of 595 nm. A combination index (CI) was dertermined using the data obtained from MTT assay with CompuSyn software [29]. CI values indicate a synergistic effect when < 1, an antagonistic effect when > 1, and an additive effect when equal to 1. Half-maximal inhibitory concentrations (IC₅₀) were determined by the GraphPad Prism software 7.0 (USA).