Mtt Assay Protocol

The measurable signal originates from viable cells reducing MTT through cellular reductase activity. This reaction converts the yellow tetrazolium compound into insoluble purple formazan, linking cellular metabolic activity to visible color formation. After the formazan is dissolved, its absorbance provides a quantitative signal for comparing metabolically active cultures under different experimental conditions.

Absorbance reflects the metabolic activity available to reduce MTT, not simply the number of cells present. Cultures with similar cell numbers may therefore produce different signals if their metabolic activity differs. For this reason, MTT results are most appropriately interpreted as estimates of viability or proliferation and as comparisons between experimental and untreated cultures.

The absorbance measured for a treated culture is evaluated against an untreated culture to identify changes associated with the experimental condition. A higher signal generally indicates more metabolically active cells, whereas a lower signal suggests reduced activity consistent with a cytotoxic or growth-inhibiting effect. These comparisons support analysis of drug responses and other treatments.

Cultured cells are exposed to the treatment or growth condition being tested, and MTT is then used to allow viable cells to generate purple formazan. Because the product is insoluble, it must be dissolved before measurement. Finally, absorbance is determined spectrophotometrically, typically at approximately 570 nm, and compared across cultures.

This protocol is useful when researchers need to compare how drugs, toxic substances, growth conditions, or other experimental treatments affect cultured cells. It can support studies of cell viability, proliferation, and cytotoxicity by revealing changes in metabolic activity. The method is particularly informative when treated cultures must be evaluated relative to untreated controls.

Results should be interpreted as treatment-associated differences in cellular metabolic activity rather than as an absolute measurement of cell number. Increased absorbance generally corresponds to more metabolically active cells, while decreased absorbance may indicate reduced viability or proliferation. This distinction matters when using the assay to evaluate toxicity, drug response, or altered growth conditions in biology.