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

Overview

This video describes the colorimetric assay, methyl thiazole tetrazolium (MTT) assay, to measure drug-induced cytotoxicity in cancer cells and determine their metabolic activity. This assay is based on the metabolic conversion of tetrazolium salts into colored products, reflecting the mitochondrial activity of cells.

Protocol

1. MTT Assay for Leukemic Cells

1. Prepare in advance the MTT solution: Dissolve 500 mg of MTT formazan in 10 mL PBS and stir (protected from light) with a magnetic stirrer for approximately 1 h. Sterilize the solution with a 0.22 µm filter.
   NOTE: The solution can be stored in 10 mL aliquots at −20 °C. Protect from direct light after thawing.
2. Prepare in advance the acidified isopropanol: Add 50 mL of 2 M HCl to 2.5 L of isopropanol.
   NOTE: Store the solution for at least one month at room temperature before use. If the isopropanol is not acidified correctly, it might form precipitates with the medium and compromise the spectrophotometric readout.
3. Prepare a separate 96-well flat bottom "Day 0" (control) plate to ensure more accurate estimation of growth inhibition: Dedicate 3 to 6 wells per cell line, add 30 µL of growth medium and 120 µL of cell suspension (8,000 cells) per each well and 150 µL of growth medium to wells corresponding to blanks (no cells). Proceed from step 1.9 to step 1.13 of this section to measure the optical density (OD).
4. Prepare a 96-well flat-bottom experimental plate: Dedicate 30 wells to drug concentrations (10 concentrations, each in triplicate), 10 wells to control cells, and 10 wells to control medium without cells (blanks) and prepare a drug dilution range of dexamethasone (Dex) using Dimethyl Sulfoxide as a solvent.
   NOTE: For CEM-WT cells the Dex dilution range is between 2 µM and 0.97 nM. For CEM/R30dm, CEM-R5 and CEM-C3 cells the Dex dilution range is between 640 µM and 0.33 nM.
5. Add 30 µL from each Dex dilution into an appropriate well of the 96-well plate. Make sure to include each concentration in a triplicate.
6. Add 30 µL of growth medium to wells corresponding to control cells and 150 µL of growth medium to wells corresponding to blanks.
7. Harvest exponentially growing cells and resuspend at their optimal seeding concentration.
   NOTE: In order to determine optimal starting cell concentrations, it is recommended to assess the growth profile of each cell line in a 96-well plate by seeding cells at several concentrations and measuring it daily for at least 4 days. Choose a seeding concentration that prevents overgrowth of cells after 72 h, as this will influence the experiment by saturating the OD values. For CEM-WT, CEM-C5, and CEM-R5, the optimal seeding concentration is 8,000 cells/well, while for CEM/R30dm, it is 5,000 cells/well.
8. Add 120 µL of the cell suspension to each well containing either the drug solution or the growth medium (wells corresponding to control cells). Fill the empty outer wells of the plate with 150 µL PBS to ensure good humidity in the plate and incubate the plates for 72 h at 37 °C with 5% CO₂ in a cell culture incubator.
9. Add 15 µL of the MTT solution to each well and shake the plate for 5 min with a plate-shaker up to a maximum of 900 shakes/min.
10. Place the plates back at 37 °C with 5% CO₂ in a cell culture incubator and incubate for another 4–6 h.
11. Add 150 µL of the acidified isopropanol to each well and mix well with a multichannel pipette to thoroughly resuspend all the formazan crystals. Start with the blank wells and make sure to rinse the tips well before proceeding to another row of the plate.
12. Incubate the plate at room temperature (RT) for 10 min.
13. Using a microplate reader, determine the OD at 540 and 720 nm to ensure an accurate measurement by correcting for background OD. Then, save data in a spreadsheet file and analyze it.

2. Data Analysis for MTT Assay

1. Calculate the OD values of cells at "Day 0" using the following formula:
   OD_{Day 0} = OD_{control cells} - Average OD_{blank wells}
2. Calculate the percentage of surviving cells for each drug concentration according to the following formula:
   % Treated cells = Average [OD_{treated cells} - Average OD_{blank wells} - OD_{Day 0}]/[OD_{control cells} - Average OD_{blank wells} - OD_Day0]*100
3. Plot the dose-response curve (drug concentration vs. growth inhibition in %).
4. Calculate the concentration of the drug that inhibits the growth of cells by 50% (IC50) using the dose-response curve.

Materials

Name	Company	Catalog Number	Comments
CCRF-CEM	ATCC, Manassas, VA, USA	ATCC CCL-119
RPMI-1640	Gibco, Carlsbad, CA, USA	11875093
Fetal bovine and calf serum	Greiner Bio-One, Frickenhausen, Germany	758093
penicillin G streptomycin sulphate	Gibco, Carlsbad, CA, USA	15140122
Tris(hydroxymethyl)-aminomethane	Sigma Aldrich	252859
CELLSTAR Cell Culture Flasks 25 centrimeter square	Greiner Bio-One, Frickenhausen, Germany	82051-074
MTT formazan	Sigma Aldrich	M2003
Anthos-Elisa-reader 2001	Labtec, Heerhugowaard, Netherlands		UV-Vis 96-well plate spectrophotometer
Greiner CELLSTAR 96 well plates	tes Greiner/Sigma	M0812-100EA
Phosphate Buffered Saline (NaCl 0.9%)	B.Braun Melsungen AG, Germany	362 3140
Trypsin/EDTA Solution 100 ml	Lonza, Basel, Switzerland	CC-5012