Cell-based quantitative evaluation of the MTT assay

OBJECTIVE: To analyze the bioreduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) on a per cell basis and evaluate its modulation as a function of different stages of cell metabolism. STUDY DESIGN: Following MTT bioreduction, total optical density (TOD), cell area and specific activity (TOD/area) of V79 cells and cultured macrophages were recorded for individual cells by means of digital image analysis. The effect of different serum (0-10% vol/vol) or genistein (0-100 microM) concentrations was used to modulate the MTT-specific activity response. RESULTS: As cells in culture are heterogeneous in cell size, the contribution of each cell to the total amount of formazan formed per dish is variable. The production of formazan per cell as a result of MTT bioreduction was found to be proportional to cell size. CONCLUSION: Specific MTT-reducing activity was analyzed in phagocytes and nonphagocyte cells, revealing the utility of this variable in evaluating the MTT assay at the single-cell level.