MTT assay

Human colon cancer cells HCT-116 and HT-29 were plated in 96-well microtiter plates with 6000 cells/well and allowed to attach for 24 h at 37 °C. The test compounds (in DMSO) were added to cell culture medium to the desired final concentrations (final DMSO concentrations for control and treatments were 0.1%). After the cells were cultured for 48 h, the medium was aspirated and cells were treated with 200 μL fresh medium containing 2.41 mmol/L 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumomide (MTT). After incubation for 3 h at 37 °C, the medium containing MTT was aspirated, 100 μL of DMSO was added to solubilize the formazan precipitate, and plates were shaken gently for 1 h at room temperature. Absorbance values were derived from the plate reading at 550 nm on a Biotek microtiter plate reader (Winooski, VT). The reading reflected the number of viable cells and was expressed as a percentage of viable cells in the control. Both HCT-116 and HT-29 cells were cultured in McCoy’s 5 A medium. The media used above was supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% glutamine, and the cells were kept in a 37 °C incubator with 95% humidity and 5% CO₂. The IC₅₀ values were conducted by using GraphPad Prism software (version 5).