Cell-based quantitative evaluation of the MTT assay

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. Following MTT bioreduction, total optical density (TOD), cell area and specific activity (TOD/area)...

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Published inAnalytical and quantitative cytology and histology Vol. 25; no. 5; p. 254
Main Authors Molinari, Beatriz L, Tasat, Débora R, Palmieri, Mónica A, O'Connor, Silvia E, Cabrini, Rómulo L
Format Journal Article
LanguageEnglish
Published United States 01.10.2003
Subjects
Animals
Coloring Agents
Connective Tissue Cells - cytology
Connective Tissue Cells - metabolism
Connective Tissue Cells - ultrastructure
Cricetinae
Cytodiagnosis - methods
Cytological Techniques - methods
Diagnostic Imaging - methods
Endosomes - metabolism
Fibroblasts - cytology
Fibroblasts - metabolism
Fibroblasts - ultrastructure
Image Processing, Computer-Assisted - methods
Macrophages, Alveolar - cytology
Macrophages, Alveolar - metabolism
Macrophages, Alveolar - ultrastructure
Mice
Mice, Inbred Strains
Microscopy, Confocal
Oxidation-Reduction
Tetrazolium Salts - metabolism
Tetrazolium Salts - pharmacokinetics
Thiazoles - blood
Thiazoles - metabolism
Thiazoles - pharmacokinetics
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Summary: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. 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. 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. 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.
ISSN:0884-6812