MTT Assay

What is MTT?

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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. ⁽⁹⁾

 

What is the principle of the MTT assay?

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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. ⁽³⁾

 

Is MTT cell-permeable?

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Yes, 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. ⁽⁶⁾

Is MTT toxic to cells?

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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. ⁽²⁾

Is MTT water soluble

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

How do I make an MTT stock solution?

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MTT is generally supplied as a lyophilized solid and 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. ⁽²⁾

How do I do an MTT assay?

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The MTT assay is a colorimetric assay for measuring the number of viable cells in culture. It relies on the intracellular reduction of MTT to formazan by mitochondrial dehydrogenases. MTT is typically prepared in PBS, then added directly to cells in culture and incubated for 1-4 hours. Live, metabolically active cells reduce yellow MTT to insoluble purple formazan crystals, which accumulate in both the cells and medium. A solubilzation solution (e.g., DMSO, acidified ethanol solution, or SDS diluted in HCl) is added to dissolve the formazan crystals and the absorbance of the solution is quantified using a spectrophotometer. The amount of formazan produced is directly proportional to the number of viable cells. ^{(4), (7)}

Sample MTT Assay Protocol:

1. Prepare cells and test compounds in 96-well plates containing a final volume of 100 µl/well. For 384-well plates, a volume of 25 µL/well is recommended
2. Incubate for desired period of exposure (e.g., 24, 48, or 96 hours) in a 37°C, 5% CO₂ incubator.
3. For a 96-well plate, add 140 µl MTT working solution to each well. For a 384-well plate, add 35 µL/well.
4. Incubate 1 to 4 hours at 37°C, protected from light. Depending on the individual cell type and concentration used, longer incubation times may be necessary.
5. Add 100 µl Solubilization solution to each well to dissolve formazan crystals.
6. Mix to ensure complete solubilization.
7. Record absorbance at 570 nm.

What is a blank in an MTT assay?

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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. ⁽¹⁰⁾

What is a positive and negative control in an MTT assay?

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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%. ⁽¹⁾

What is the optical density (OD) range in an MTT assay?

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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. ⁽⁵⁾

How do I calculate cell viability in MTT assay?

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In an MTT assay, the percentage of cell viability is calculated using the following equation: ⁽⁸⁾

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

References

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1. 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
2. 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
3. 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
4. Plumb J. A. (1999). Cell sensitivity assays : the MTT assay. Methods in molecular medicine, 28, 25–30. https://doi.org/10.1385/1-59259-687-8:25
5. 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/
6. 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/
7. Sylvester P. W. (2011). Optimization of the tetrazolium dye (MTT) colorimetric assay for cellular growth and viability. Methods in molecular biology (Clifton, N.J.), 716, 157–168. https://doi.org/10.1007/978-1-61779-012-6_9
8. 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
9. 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
10. 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