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