MTT/MTS Assay
MTT/MTS Cell Viability and Proliferation Assay · Also known as: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, tetrazolium assay, mitochondrial activity assay
The MTT assay, introduced by Tatsuro Mosmann in 1983, is a colorimetric method for quantifying cell viability and proliferation by measuring mitochondrial metabolic activity. The method detects the conversion of the water-soluble tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by active mitochondria, producing an insoluble purple formazan precipitate proportional to the number of viable cells. The related MTS assay, which does not require solubilization, offers improved kinetics and is now widely adopted in both academic research and pharmaceutical development.
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When to use it
The MTT assay is ideal for screening drug toxicity, assessing the biocompatibility of biomaterials and scaffolds, and measuring cell proliferation in response to growth factors or culture conditions. It is preferred for endpoint assays where a single measurement at a defined timepoint is acceptable. However, the assay is destructive (cells cannot be recovered for further analysis), has limited kinetic information, and can produce false positives if cells are metabolically inactive but not dead. For real-time monitoring or kinetic studies, consider label-free impedance-based methods or fluorescent live-cell assays. MTS, as a soluble variant, is preferred when faster kinetics or reduced handling are important.
Strengths & limitations
- Simple, rapid, and cost-effective: the assay can be completed in hours with minimal equipment beyond a microplate reader.
- Quantitative and sensitive: can detect changes in cell number over a range of 10²–10⁵ cells per well.
- High-throughput capable: 96-well or 384-well formats enable screening of hundreds of conditions in parallel.
- Endpoint or kinetic variants: MTT is endpoint only, but MTS allows kinetic monitoring without cell lysis.
- Widely standardized: the assay is well-established in regulatory testing and pharmaceutical development, with extensive precedent for interpretation.
- Endpoint assay: cells are lysed and destroyed, precluding subsequent analysis or recovery of living cells.
- Slow kinetics for MTT: the precipitation step requires several hours of incubation and solubilization; MTS overcomes this but is slightly more expensive.
- Limited information about cell health: metabolic activity does not fully capture cell state; apoptotic cells may retain mitochondrial function temporarily.
- Interference with colored compounds: some test compounds (antioxidants, reducing agents) can directly reduce tetrazolium salts, causing false positives.
Frequently asked
Why are my absorbance readings not proportional to cell number?
Check for incomplete solubilization of formazan crystals—ensure adequate incubation time and thorough pipetting with the solubilization reagent. Also verify that phenol red is not present in the culture media (it interferes with 570 nm absorbance). If these are correct, the assay may be saturated at very high cell densities; use fewer cells or dilute the formazan suspension.
Can I use MTT to test a drug that is known to have antioxidant activity?
Antioxidants and reducing agents can directly reduce tetrazolium salts, giving false positives. For such compounds, use a parallel colorimetric assay that is independent of reduction chemistry, such as the LDH release assay or the live/dead staining assay. Alternatively, include positive and negative controls that are chemically related to your test compound to detect false signals.
What is the difference between MTT and MTS, and which should I use?
MTT requires a solubilization step after the assay and has slower kinetics but is less expensive. MTS is water-soluble, requires no solubilization, and allows kinetic readings over time; it is preferred for kinetic assays or when handling speed is critical. For endpoint assays, either is acceptable; choice depends on budget and available equipment.
How do I know if my cells are truly dead or just metabolically quiescent?
MTT measures only metabolic activity, not viability per se. To distinguish death from quiescence, use complementary assays: measure membrane integrity with trypan blue exclusion, perform flow cytometry with propidium iodide staining, or use live/dead fluorescent dyes. Combining methods provides a more complete picture of cell status.
Can I freeze my plates and run the assay later?
Yes, cells can often be frozen after plating or after MTT incubation (but before solubilization); however, freezing can damage some cell types. If you freeze cells, use a cryoprotectant (e.g., DMSO) and thaw carefully. It is preferable to complete the assay fresh to avoid variability.
Sources
- Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65(1-2), 55-63. DOI: 10.1016/0022-1759(83)90303-4 ↗
- Slade, P. G. (1999). MTS tetrazolium compound (abstract). Methods in Cell Biology, 63, 65-72. link ↗
- Riss, T. L., Moravec, R. A., Niles, A. L., et al. (2004). Cell viability assays. In Assay Guidance Manual. Eli Lilly & Company and the National Center for Advancing Translational Sciences. link ↗
How to cite this page
ScholarGate. (2026, June 3). MTT/MTS Cell Viability and Proliferation Assay. ScholarGate. https://scholargate.app/en/biomaterials/mtt-mts-assay
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