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

Abstract

Source: Sciarrillo, R. et al. Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models. J. Vis. Exp. (2016)

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 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. Prepare in advance the acidified isopropanol: Add 50 mL of 2 M HCl to 2.5 L of isopropanol. <stron…

Materials

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