MTT colorimetric assay for testing macrophage cytotoxic activity in vitro

Abstract

The MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) cleavage assay, originally described by Mosmann (1983, J. Immunol. Methods 65, 55) for measuring cell survival/proliferation, has been used successfully to quantitate macrophage-mediated cytotoxicity. Peritoneal macrophages, from control or pyran copolymer MVE2-treated mice and from TU5 and L929 murine cell lines, were used as effectors and targets respectively in a 48 h cytotoxicity test, at three effector: target ratios (10:1, 5:1, 2:1). The amount of MTT reduced by cells to its blue formazan derivative during an additional 4 h of culture was quantified spectrophotometrically at 570 nm using an ELISA reader. A linear relationship between the formazan generated and the number of viable TU5 and L929 cells was demonstrated, together with time-dependent growth characteristics for these cells. The formazan produced by macrophages was independent of their functional state and did not interfere with the target cell signal. MVE2-activated macrophages strongly inhibited the survival/growth of target cells in a dose-related way, whereas the cytotoxic activity of control macrophages was very low. Finally, the MTT method compared favorably with the 3H-TdR uptake method in evaluating macrophage cytotoxicity, and both of them were more sensitive than the 3H-TdR release assay. The MTT cleavage method is a useful alternative to radioisotopic methods for quantitating macrophage cytotoxicity for actively growing in vitro targets. Its main advantages are: (a) sensitivity and reproducibility; (b) elimination of the need for radioactive compounds; (c) ease with which it can be performed and quantified; (d) rapidity.