The 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide （MTT） assay is a routine cell viability assay for cell proliferation and cytotoxicity, which is widely used in many fields, especially in screening for drug discovery. However, this assay exhibits limitations in the presence of particular compounds and under certain assay conditions, which may yield false screening results. For example, polyphenols that are extracted from natural sources can react with MTT in the absence of living cells and thus interfere with the screening results. We measured the absorbance of 15 polyphenols extracted from green tea and showed that the phenolic hydroxyl groups in the polyphenols are responsible for the reduction of MTT to formazan. When three or more phenolic hydroxyl groups were present on a conjugated polyphenol, a significantly increased MTT reduction was observed. Moreover, the type of medium also had an effect on the absorbance value, in the following order： ct-MEM ＋ 10% FBS〉 a-MEM〉DMEM/F12〉PBS. The absorbance of the MTT assay recorded at 570 nm is more sensitive than that measured at 595 nm. These results will improve the cell-based assay of polyphenols and clarify the limitations of the MTT assay as a method of screening in drug discovery.
