MTT Assay Calculator

Additional Information

The MTT assay works on the principle of enzymatic reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by viable cells to form purple formazan crystals. The general procedure for this assay involves treating cultured cells in a well plate with various compounds or experimental conditions, adding MTT solution to the cells, and incubating them to allow the reduction of MTT by mitochondrial enzymes. After incubation, the formazan crystals produced are dissolved in a solvent such as dimethyl sulfoxide (DMSO), and the resulting solution is measured spectrophotometrically at a specific wavelength. The absorbance values obtained reflect the amount of formazan produced, which is proportional to the number of viable cells. A higher concentration of formazan crystals and a more intense purple color indicate higher cellular metabolic activity and thus a larger population of viable cells; however, it is important to note that because this assay provides only a relative measure of cell viability and proliferation, results should be interpreted within the context of each assay's experimental design and treatment groups.

When analyzing the results of an MTT assay, absorbance values of sample wells are compared against those of control wells in order to determine the relative viabilities of cells from the sample. Control wells contain live, untreated cells with implied relative viabilities of approximately 100%, and the viabilities of the sample cells are reported as either percentages or decimals of the control. Viabilities of the control wells may differ slightly due to variations in their measured absorbance values; however, the mean viability of the control column will always amount to 100%. For this tool, the viability of each well is determined by the formula:

In a typical MTT assay, wells are split up into columns, where each column contains wells with the same number of cells which are given the same treatment. The mean absorbance of each column is then taken to minimize the effect of errors or random outliers from a single well. For this reason, this tool also provides the mean viability of each column as a way to represent the average viability of cells from each treatment.

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