Interference of Antioxidant Flavonoids with MTT Tetrazolium Assay in a Cell Free System

Abstract

Introduction: The tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) used extensively to measure the quantitative survival and proliferation of mammalian cells. The analysis is based upon the reduction of MTT by metabolically active cells to insoluble formazan crystals. Flavonoids are a large group of natural compounds found in plants with variable phenolic structures.  Flavonoids, as they are potential reducing agents, they act as a free radical scavenger. The aim of the study is to assess the reducing effect of some of the flavonoids on tetrazolium salt and their interference with the colorimetric analysis of MTT. The cell viability obtained from the MTT assay was compared with that of SRB assay in the determination of flavonoids cytotoxicity.  Materials and Methods: The present study examined the effect of few bio-flavonoids like Quercetin, EGCG, Rutin and Resveratrol to reduce MTT in the absence of cells under different experimental conditions such as concentration of flavonoids, incubation time and results were compared with SRB assay findings. The study also involves the analysis of flavonoid cytotoxicity on lung cancer cells NCIH-460 and NCIH-522 by MTT and SRB assay to establish the suitable cell viability assay for  flavonoids. Results: All the flavonoids showed the instant formation of the dark blue formazan salt in the absence of the cells with MTT assay. Whereas SRB assay of flavonoids in the absence of cells, results showed the absorbance similar to that of the blank, indicating that SRB did not interfere with flavonoids in a cell-free system. Conclusion: From the results, it is evident that MTT is not a suitable method to determine the effect of flavonoids on cell viability and proliferation as flavonoids itself reduces the MTT to formazan crystals. Study also suggests that SRB assay is more suitable method to determine the effect of flavonoids on cell viability.