Methyl Thiazolyl Tetrazolium or MTT Assay: A Colorimetric Assay to Measure Drug Resistance via Determination of Metabolic Activity in Cancer Cells

Methyl thiazole tetrazolium or MTT assay allows cell viability assessment based on the measurement of cellular metabolic activity.

To begin the assay, prepare a range of increasing concentrations of the drug of interest. Add equal volumes of the different drug concentrations into the wells of a multi-well plate.

Next, pipette a suspension of desired drug-sensitive parental cancer cells and their drug-resistant variants in a suitable media, each into a set of wells containing different drug concentrations. Incubate the plate.

In culture, the drug-sensitive cancer cells are sensitive to the drug molecules, leading to cell death even at low concentrations. On the contrary, the drug-resistant cancer cells are insensitive to the drug molecules, facilitating their survival and proliferation even at higher drug concentrations.

Treat the cells with MTT solution and incubate. MTT, a positively charged tetrazolium salt, readily penetrates the metabolically active cells. Within these cells, NAD(P)H-dependent oxidoreductase enzymes reduce the MTT salt to water-insoluble formazan crystals, which get transported to the cell surface. 

Now, add a suitable solubilization solution into the wells. Mix well to dissolve the formazan crystals into a colored solution. Finally, use a microplate reader to measure the absorbance of the colored solution, indicative of cellular metabolic activity.

The wells containing drug-resistant cancer cells display higher color intensity, suggesting increased cell viability compared to the drug-sensitive cancer cells.