Cell Viability Evaluation by MTT Assay

Cell viability was analysed by 3-(4, 5-dimeth-ylthiazol-2-yl)-5-(3-carboxymethxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTT) assay as previously described (15 (link)). Cell suspensions (500 µl) from the RKO-PBS and RKO-Nitrodi cells, containing 3×10⁴ viable cells, were plated in 24 multi-well plates. To measure MTT reduction by colorimetric assay, the cells were washed and incubated for 3 h in 100 µl DMEM without phenol red (D2429, Sigma-Aldrich; Merck KGaA), and supplemented with 0.45 mg/ml MTT. The medium was then replaced by 100 µl 0.1 M HCl in isopropanol and the cells were incubated at 37°C for 30 min for lysis. The insoluble formazan was resuspended and optical densities were measured at a wavelength of 570 nm using a microplate reader (BioTek Synergy Microplate Reader; BioTek Instruments, Inc.), according to the MTT manufacturer's protocol. The results represent the mean of 3 experiments, each performed in duplicate.