MTT Assay | AAT Bioquest

Cell Meter™ Colorimetric MTT Cell Proliferation Kit

The MTT assay is a colorimetric cell viability test that quantifies metabolically active cells through the reduction of yellow MTT tetrazolium salt to purple formazan crystals, measurable at 550-570nm.

Colorimetric Quantification: Yellow-to-purple color change measured at 550-570nm, directly proportional to viable cell count
Cell-Permeable Design: Positive charge allows MTT to cross intact plasma membranes, unlike negatively charged alternatives (MTS, XTT, WST-1)
Non-Radioactive Safety: Eliminates health hazards and disposal costs of radioactive 3H-thymidine assays while maintaining sensitivity
Metabolic Activity Detection: Measures mitochondrial succinate dehydrogenase activity as a direct indicator of cell viability

Example of an MTT assay performed in a 96-well microplate. As the intenisty of the purple signal decreases from lane 1 to lane 12, so decreases the number of viable metabolically active cells per well.

Protocol

COA

Catalog	Size	Price	Quantity
22768	1000 Tests	Price
22769	5000 Tests	Price

Overview

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

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
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
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/
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/
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

References

View all 50 references: Citation Explorer

Novel Dental Poly (Methyl Methacrylate) Containing Phytoncide for Antifungal Effect and Inhibition of Oral Multispecies Biofilm.

Authors:

Lee, Myung-Jin and Kim, Min-Ji and Oh, Sang-Hwan and Kwon, Jae-Sung

Journal:

Materials (Basel, Switzerland) (2020)

Toxicity of diuron metabolites in human cells.

Authors:

Mohammed, Ali Mustafa and Huovinen, Marjo and Vähäkangas, Kirsi H

Journal:

Environmental toxicology and pharmacology (2020): 103409

[Comparative study on quality of decoction pieces of Saposhnikovia divaricata with different growth patterns and years and thinking of standard of decoction pieces of S. divaricata in Chinese Pharmacopoeia].

Authors:

Xue, Xue and Wang, Hao and Jia, Tian-Ying and Qu, Wen-Jia and Wang, Hai-Li and Xin, Jie-Ping and Liu, Meng-Nan and Xiong, Hui and Li, Xiang-Ri

Journal:

Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica (2019): 4034-4042

Evaluation of hepatotoxicity potential of a potent traditional Tibetan medicine Zuotai.

Authors:

Zhou, Liang-Liang and Chen, Hai-Juan and He, Qiang-Qiang and Li, Cen and Wei, Li-Xin and Shang, Jing

Journal:

Journal of ethnopharmacology (2019): 112-118

Development and validation of UPLC method for WST-1 cell viability assay and its application to MCTT HCE™ eye irritation test for colorful substances.

Authors:

Joo, Kyung-Mi and Kim, Seolyeong and Koo, Ye Ji and Lee, Miri and Lee, Su-Hyun and Choi, Dalwoong and Lim, Kyung-Min

Journal:

Toxicology in vitro : an international journal published in association with BIBRA (2019): 412-419

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12171501

Instrument settings

Absorbance microplate reader
Absorbance	562 nm
Recommended plate	Clear bottom

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Page updated on October 11, 2025

Component A: MTT Reagent A	40 mL
Component B: MTT Reagent B	100 mL

Component A: MTT Reagent A	2 X 100 mL
Component B: MTT Reagent B	500 mL