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.
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