MTT Assay

The MTT assay is a quantitative test which measures cell viability by observing the color change of the MTT substrate from yellow to purple.

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a yellow tetrazolium salt commonly used to measure cell viability, proliferation, and cytotoxicity. In live metabolically active cells, water-soluble MTT is reduced in the mitochondria by succinate dehydrogenase to insoluble purple formazan crystals. The amount of formazan produced can be quantified spectrophotometrically (550 nm) upon solubilization and is directly proportional to the number of viable cells in the culture.

The MTT assay is a safe, sensitive, and reliable indicator of cell viability and is preferred over other endpoint viability assays, such as the radioactive 3H-thymidine incorporation assay. Besides having a lengthy and tedious protocol, the health hazards of working with radioactive materials are well recognized, as are the accompanying economic considerations required for the appropriate handling and disposal of radioactive waste. ^[5]

Principle and mechanism

The principle behind the MTT assay is that only viable cells have active metabolism and can convert MTT to purple formazan, which has an absorbance maximum at 570nm. The amount of formazan produced can be measured using a spectrophotometer and is proportional to the number of viable cells.

When cells die, they lose their metabolic activity and are unable to convert MTT resulting in no visible color change. ^[2]

Permeability

MTT is cell-permeable. The net positive charge of MTT allows it to readily penetrate the intact plasma membranes of viable cells. Other tetrazolium salts, such as MTS, XTT, and WST-1, are negatively charged compounds and unable to cross the plasma membrane. ^[4]

Cytotoxicity

Since MTT is a salt, high levels may be toxic to cells. Should the stock concentration of MTT be too high, cells may be disturbed and reduce viability readings. In such high concentrations, the cytotoxicity of formazan can also pose experimental difficulties when the culture media must be removed from the well-plate. ^[1]

Solubility

MTT is soluble in water (10 mg/mL), ethanol (20 mg/mL), buffer salt solutions (5 mg/mL), and culture media (5 mg/mL). ^[3]

References

1. Grela, E., Kozłowska, J., & Grabowiecka, A. (2018). Current methodology of MTT assay in bacteria - A review. Acta histochemica, 120(4), 303–311. https://doi.org/10.1016/j.acthis.2018.03.007
2. Kumar, P., Nagarajan, A., & Uchil, P. D. (2018). Analysis of Cell Viability by the MTT Assay. Cold Spring Harbor protocols, 2018(6), 10.1101/pdb.prot095505. https://doi.org/10.1101/pdb.prot095505
3. Plumb, J. A., Milroy, R., & Kaye, S. B. (1989). Effects of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer research, 49(16), 4435–4440. https://pubmed.ncbi.nlm.nih.gov/2743332/
4. Riss, T. L., Moravec, R. A., Niles, A. L., Duellman, S., Benink, H. A., Worzella, T. J., & Minor, L. (2013). Cell Viability Assays. In S. Markossian (Eds.) et. al., Assay Guidance Manual. Eli Lilly & Company and the National Center for Advancing Translational Sciences. https://www.ncbi.nlm.nih.gov/books/NBK144065/
5. van Tonder, A., Joubert, A. M., & Cromarty, A. D. (2015). Limitations of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays. BMC research notes, 8, 47. https://doi.org/10.1186/s13104-015-1000-8

In an MTT assay, the percentage of cell viability is calculated using the following equation:

% Viability = ( Mean OD_sample / Mean OD_blank ) x 100

van Meerloo, J., Kaspers, G. J., & Cloos, J. (2011). Cell sensitivity assays: the MTT assay. Methods in molecular biology (Clifton, N.J.), 731, 237–245. https://doi.org/10.1007/978-1-61779-080-5_20

The blank in the MTT assay is a cell-free medium plus an MTT solution and a solubilizing buffer. The blank represents the background absorbance values created by the medium itself that must be subtracted from final values of the samples, standard curves, and positive and negative controls. Without addition of the blanks, and subsequent subtraction of values from test results, MTT assay analysis will not be accurate.

Yang, Y., Lu, Y., Wu, Q. Y., Hu, H. Y., Chen, Y. H., & Liu, W. L. (2015). Evidence of ATP assay as an appropriate alternative of MTT assay for cytotoxicity of secondary effluents from WWTPs. Ecotoxicology and environmental safety, 122, 490–496. https://doi.org/10.1016/j.ecoenv.2015.09.006

In an MTT assay the positive control wells will contain untreated cells, MTT solution and a solubilizing buffer. Viability will be close to 100%. Negative control wells will contain only dead cells, MTT solution and a solubilizing buffer. Viability will be 0%.

Bae, S. H., Che, J. H., Seo, J. M., Jeong, J., Kim, E. T., Lee, S. W., Koo, K. I., Suaning, G. J., Lovell, N. H., Cho, D. I., Kim, S. J., & Chung, H. (2012). In vitro biocompatibility of various polymer-based microelectrode arrays for retinal prosthesis. Investigative ophthalmology & visual science, 53(6), 2653–2657. https://doi.org/10.1167/iovs.11-9341

Instead of measuring the absorbance for MTT, what is really needed is the maximum absorbance of formazan. The range of absorbance for formazan is between 400-650 nm, with a peak between 540-570 nm. In most MTT assay protocols, it is recommended to read absorbance at OD = 590 nm.

Plumb, J. A., Milroy, R., & Kaye, S. B. (1989). Effects of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer research, 49(16), 4435–4440. https://pubmed.ncbi.nlm.nih.gov/2743332/

DMSO is added to MTT assays at the end of the experiment to dissolve the formazan crystals that were created during the experiment. If DMSO is not added and the formazan crystals are not dissolved, this would cause significant changes in the absorbance spectrum due to the sodium bicarbonate that would still be there.

Twentyman, P. R., & Luscombe, M. (1987). A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. British journal of cancer, 56(3), 279–285. https://doi.org/10.1038/bjc.1987.190

From powder

If MTT is supplied as a lyophilized solid, it must be resuspended in a solution, such as PBS, before loading into cells. We recommend using a 12 mM stock solution. To prepare a 12 mM MTT stock solution, dissolve 5 mg of MTT in 1 mL PBS. This stock solution is enough for 100 reactions (10 µL per reaction) and can be stored at 4 °C protected from light for up to 4 weeks.

For AAT Bioquest kit #22768 and #22769

Prepare the amount of MTT working solution needed by mixing 4 mL of MTT Reagent A (Component A) with 10 mL of MTT Reagent B (Component B) (2:5, v/v ratio of MTT Reagent A : B). Mix well. Note that 14 mL MTT working solution is enough for 100 tests in a 96-well plate. Prepare enough MTT working solution right before the experiment, use promptly.

Grela, E., Kozłowska, J., & Grabowiecka, A. (2018). Current methodology of MTT assay in bacteria - A review. Acta histochemica, 120(4), 303–311. https://doi.org/10.1016/j.acthis.2018.03.007