Assays which allow for the quantitative measurement of cell death during cell culture are crucial to any experiment involving cell lines or ex vivo cellular clinical samples. In pre-clinical drug discovery, potential drug candidates are usually tested against mammalian cell lines such CHO or Vero in order to assess any cytotoxic effects the compound could exert on the body’s own cells. In more fundamental research where the effects of perturbation factors are tested on cellular phenotype, gene, or protein expression, the cytotoxic effects of the perturbing factor must first be investigated via a cell viability assay.
The viability assay most commonly used throughout the world is the MTT assay, first described by Tim Mosmann in 1983. This colorimetric assay uses reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, or MTT) to measure cellular metabolic activity as a proxy for cell viability. Viable cells contain NAD(P)H-dependent oxidoreductase enzymes which reduce the MTT reagent to formazan, an insoluble crystalline product with a deep purple color. Formazan crystals are then dissolved using a solubilizing solution and absorbance is measured at 500-600 nanometers using a plate-reader. The darker the solution, the greater the number of viable, metabolically active cells.
Performing an MTT assay is easy enough, but there can be pitfalls if one is unfamiliar with the protocol. Below is a brief description of the steps:
Interpreting results
The absorbance reading of the blank must be subtracted from all samples. Absorbance readings from test samples must then be divided by those of the control and multiplied by 100 to give percentage cell viability or proliferation (see formula below). Absorbance values greater than the control indicate cell proliferation, while lower values suggest cell death or inhibition of proliferation.
Tips and troubleshooting
Reagents
Absorbance values
General tips
Limitations of the MTT assay
The sensitivity of an MTT assay is lower than that of fluorescent or luminescent assays, particularly with cells that do not readily proliferate or cells with low metabolic activity. Furthermore, certain chemical compounds interfere with the reduction of MTT to formazan and are therefore not easily compatible with MTT assays. These include polyphenols, vitamin A, coenzyme A, and DTT (dithiothreitol). It should also be noted that both exposure to MTT and formation of formazan crystals are cytotoxic and cause damaging changes to cellular morphology.
If performed correctly, an MTT assay is an extremely useful part to many experiments. However, it may not always be the best choice for your particular needs. The next article in this series will details alternatives to the MTT assay.
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