A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function.
Microculture tetrazolium assays (MTAs) are being widely applied to probe the relationships between cell survival, growth, and differentiation and also to investigate associations between compromised cell metabolism, oxidative stress, and programmed cell death as occurs in apoptosis. MTAs rely upon the cellular reduction of tetrazolium salts to their intensely coloured formazans. The resulting colorimetric assays form the basis of exceptionally precise systems which are technically amenable and capable of a high throughput of samples. As a consequence, MTAs are being used to monitor responses to both extracellular activators and toxic agents in disciplines as diverse as radiobiology and endocrinology. We review the chemistry and histochemical applications of tetrazolium salts and subsequently discuss the criteria for their use in MTAs. These assays are one of the latest examples of the application of the tetrazolium/formazan system to cell biology. We outline current views on the mechanisms of the bioreduction of tetrazolium salts. These probably combine to reflect the integrated pyridine nucleotide dependent redox state of the cell. We try to illustrate how an understanding of these mechanisms helps to avoid some of the pitfalls of the MTA systems. There is now for example, extensive evidence that changes in cell culture environments, such as glucose supply or pH of the medium, influence the reduction of tetrazolium salts and thereby introduce artefacts into MTAs. Finally, we provide examples of situations in which MTAs can be used to complement other more established experimental systems. They then act as unique probes with which to investigate changes in the redox state of the cell. These changes are associated with regulation of cell growth, proliferation and differentiation and conversely, the different pathways leading to cell death.
Marshall
N J
NJ
Department of Molecular Pathology, University College London Medical School, UK.
Goodwin
C J
CJ
Holt
S J
SJ
D000818:Animals; D002455:Cell Division; D002468:Cell Physiological Phenomena; D002478:Cells, Cultured; D003124:Colorimetry / Q000379:methods / Q000706:statistics & numerical data; D005562:Formazans / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D013778:Tetrazolium Salts
D005562:Formazans; D013778:Tetrazolium Salts