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MTT Assay of Cell Numbers after Drug/Toxin Treatment   

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MTT assay is a colorimetric method for measuring the activity of enzymes in living cells that reduce MTT to formazan dyes, giving a purple color. It is commonly used to determine cytotoxicity of potential medicinal agents and toxic materials, since these types of materials are expected to stimulate or inhibit cell viability and growth. Here, a general protocol is described to carry out an MTT assay on different types of cells.

Keywords: MTT assay, Cell numbers, Drug

Materials and Reagents

  1. Raw264.7, MCF-7 or Hela cells
  2. Thiazolyl blue tetrazolium bromide (MTT) (Sigma-Aldrich, catalog number: M5655 )
  3. DMSO
  4. DPBS (Life Technologies, InvitrogenTM, catalog number: 14190-250 )
  5. General chemicals (Sigma-Aldrich)


  1. 96 well plate
  2. Shaking table
  3. Paper towels
  4. Incubator


  1. Plate 500-10,000 cells in 200 μl media per well in a 96 well plate. Leave 8 wells empty for blank controls.
  2. Incubate (37 °C, 5% CO2) overnight to allow the cells to attach to the wells.
  3. Add 2 μl of drug of interest dissolved in DMSO to each well. Place on a shaking table, 150 rpm for 5 min, to thoroughly mix the samples into the media.
  4. Incubate (37 °C, 5% CO2) for 1-5 days to allow the drug/toxin to take effect.
  5. Make 2 ml or more of MTT solution per 96 well plate at 5 mg/ml in DPBS. Do not make a stock as MTT in solution is not stable long-term.
  6. Add 20 μl MTT solution to each well. Place on a shaking table, 150 rpm for 5 min, to thoroughly mix the MTT into the media.
  7. Incubate (37 °C, 5% CO2) for 1-5 h to allow the MTT to be metabolized.
  8. Dump off the media (dry plate on paper towels to remove residue if necessary).
  9. Resuspend formazan (MTT metabolic product) in 200 μl DMSO. Place on a shaking table, 150 rpm for 5 min, to thoroughly mix the formazan into the solvent.
  10. Read optical density at 560 nm and subtract background at 670 nm. Optical density should be directly correlated with cell quantity.


This work was funded by 5050 project by Hangzhou Hi-Tech District, Funding for Oversea Returnee by Hangzhou City, ZJ1000 project by Zhejiang Province. This protocol was developed in the Cohen Lab, Department of Genetics, Stanford University, CA, USA [Chen et al. (unpublished)].


  1. Hayon, T., Dvilansky, A., Shpilberg, O. and Nathan, I. (2003). Appraisal of the MTT-based assay as a useful tool for predicting drug chemosensitivity in leukemia. Leuk Lymphoma 44(11): 1957-1962.
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Chen, R. (2011). MTT Assay of Cell Numbers after Drug/Toxin Treatment. Bio-101: e51. DOI: 10.21769/BioProtoc.51.

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Yuanqing Lin
How many number of cells should be placed in each well for effective assay?
7/20/2011 5:14:19 PM Reply

It depends on the cell size, growth rate and treatment time. Usually we let the cells reach 50%-80% confluence before the treatment if the treatment time is within 1 day.

8/31/2011 4:03:58 PM

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