The MTT assay is a widely used method for assessing cell viability based on metabolic activity. This protocol outlines the steps to evaluate the sensitivity of drug-sensitive and drug-resistant cancer cells to a specific drug.
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.
Prepare a 96-well flat-bottom experimental plate. Dedicate wells to drug concentrations to control cells and to control medium without cells. Prepare a drug dilution range of dexamethasone or Dex. Add 30 microliters from each Dex dilution into an appropriate well of the 96-well plate. Make sure to include each concentration in triplicate.
Harvest exponentially growing leukemic cells and resuspend them at their optimal seeding concentration. Add 120 microliters of the cell suspension to each well containing either the drug solution or the growth medium and incubate at 37 degrees Celsius with 5% carbon dioxide for 72 hours. After the incubation step, add 15 microliters of MTT reagent to each well by using a repetitive pipette.
Shake the plate for 5 minutes with a plate-shaker up to a maximum of 900 shakes per minute. Place the plates back at 37 degrees Celsius with 5% carbon dioxide and a cell culture incubator and incubate for another 4 to 6 hours.
Following incubation, make sure to thoroughly resuspend the formazan crystals as the absorbance values depend on proper solubilization of the compound.
Add 150 microliters of the acidified isopropanol to each well and mix well with a multichannel pipette to thoroughly resuspend all the formazan crystals. Using a microplate reader, determine the optical density or OD at 540 and 720 nanometers to ensure an accurate measurement by correcting for background OD.
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