MTT assay#MTT and related tetrazolium salts

MTT, a yellow tetrazole, is reduced to purple formazan in living cells.

{{cite journal | vauthors = Mosmann T | title = Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays | journal = Journal of Immunological Methods | volume = 65 | issue = 1–2 | pages = 55–63 | date = December 1983 | pmid = 6606682 | doi = 10.1016/0022-1759(83)90303-4 }} A solubilization solution (usually either dimethyl sulfoxide, an acidified ethanol solution, or a solution of the detergent sodium dodecyl sulfate in diluted hydrochloric acid) is added to dissolve the insoluble purple formazan product into a colored solution. The absorbance of this colored solution can be quantified by measuring at a certain wavelength (usually between 500 and 600 nm) by a spectrophotometer. The degree of light absorption is dependent on the degree of formazan concentration accumulated inside the cell and on the cell surface. The greater the formazan concentration, the deeper the purple colour and thus the higher the absorbance.{{cn|date=May 2025}}

XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) has been proposed to replace MTT, yielding higher sensitivity and a higher dynamic range. The formed formazan dye is water-soluble, avoiding a final solubilization step.{{cite web | title = Why should I use XTT instead of MTT | work = [aniara.com] | publisher = ANIARA | url = http://www.aniara.com/pdf/literature/XTTvsMTT.pdf | format = PDF, 0.1 MB | access-date = 2010-11-19 | archive-date = 2018-04-06 | archive-url = https://web.archive.org/web/20180406111203/http://www.aniara.com/pdf/literature/XTTvsMTT.pdf | url-status = live }}

Water-soluble tetrazolium salts are more recent alternatives to MTT: they were developed by introducing positive or negative charges and hydroxy groups to the phenyl ring of the tetrazolium salt, or better with sulfonate groups added directly or indirectly to the phenyl ring.{{cn|date=May 2025}}

MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), in the presence of phenazine methosulfate (PMS), produces a formazan product that has an absorbance maximum at 490 nm in phosphate-buffered saline. The MTS assay is often described as a 'one-step' MTT assay, which offers the convenience of adding the reagent straight to the cell culture without the intermittent steps required in the MTT assay. However this convenience makes the MTS assay susceptible to colorimetric interference as the intermittent steps in the MTT assay remove traces of coloured compounds, whilst these remain in the microtitre plate in the one-step MTS assay. Precautions are needed to ensure accuracy when using this assay and there are strong arguments for confirming MTS results using qualitative observations under a microscope. (This, however, is prudent for all colormetric assays.)

{{cite journal | vauthors = Cory AH, Owen TC, Barltrop JA, Cory JG | title = Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture | journal = Cancer Communications | volume = 3 | issue = 7 | pages = 207–212 | date = July 1991 | pmid = 1867954 | doi = 10.3727/095535491820873191 }}

WSTs (water-soluble tetrazolium salts) are a series of other water-soluble dyes for MTT assays, developed to give different absorption spectra of the formed formazans.{{cite web | title = Water Soluble Tetrazolium Salts (WSTs) | work = [interchim.com] | publisher = Interchim | url = http://www.interchim.fr/ft/F/F98881.pdf | format = PDF, 0.4 MB | access-date = 2013-08-12 | archive-date = 2018-12-22 | archive-url = https://web.archive.org/web/20181222104239/http://www.interchim.fr/ft/F/F98881.pdf | url-status = live }} WST-1 and in particular WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium), are advantageous over MTT in that they are reduced outside cells, combined with PMS electron mediator, and yield a water-soluble formazan. Finally, WST assays (1) can be read directly (unlike MTT that needs a solubilization step), (2) give a more effective signal than MTT, and (3) decrease toxicity to cells (unlike cell-permeable MTT, and its insoluble formazan that accumulate inside cells).

Tetrazolium dye reduction is generally assumed to be dependent on NAD(P)H-dependent oxidoreductase enzymes largely in the cytosolic compartment of the cell.{{cite journal | vauthors = Berridge MV, Tan AS | title = Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction | journal = Archives of Biochemistry and Biophysics | volume = 303 | issue = 2 | pages = 474–482 | date = June 1993 | pmid = 8390225 | doi = 10.1006/abbi.1993.1311 }} Therefore, reduction of MTT and other tetrazolium dyes depends on the cellular metabolic activity due to NAD(P)H flux. Cells with a low metabolism such as thymocytes and splenocytes reduce very little MTT. In contrast, rapidly dividing cells exhibit high rates of MTT reduction. It is important to keep in mind that assay conditions can alter metabolic activity and thus tetrazolium dye reduction without affecting cell viability.{{cite journal | vauthors = Ghasemi M, Turnbull T, Sebastian S, Kempson I | title = The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis | journal = International Journal of Molecular Sciences | volume = 22 | issue = 23 | pages = 12827 | date = November 2021 | pmid = 34884632 | pmc = 8657538 | doi = 10.3390/ijms222312827 | doi-access = free }} In addition, the mechanism of reduction of tetrazolium dyes, i.e. intracellular (MTT, MTS) vs. extracellular (WST-1), will also determine the amount of product. Additionally, proof has been provided as to the spontaneous MTT reduction in lipidic cellular compartments/structures, without enzymatic catalysis involved. Nevertheless, even under this alternative paradigm, MTT assay still assesses the reduction potential of a cell (i.e. availability of reducing compounds to drive cellular energetics). As such, the final cell viability interpretation remains unchanged.

In studying the viability of cells seeded on 3D fibrous scaffolds, the thickness of the scaffolds may influence the MTT assay results.{{Cite journal| vauthors = Lyundup AV, Demchenko AG, Tenchurin TH, Krasheninnikov ME, Klabukov ID, Shepelev AD |date=2016|title=Improving the seeding effectiveness of stromal and epithelial cell cultures in biodegradable matrixes by dynamic cultivation |journal=Genes to Cells|volume=11|issue=3|pages=102–107|doi=10.5281/zenodo.1175840|issn=2313-1829}}

The optical density (OD) at 550 nm is used to calculate the percentage of viability results using the following equation:{{Cite web |title=ISO 10993-5:2009 |url=https://www.iso.org/cms/render/live/en/sites/isoorg/contents/data/standard/03/64/36406.html |access-date=2022-10-06 |website=ISO |language=en}}

:Viability % = 100 × OD550e / OD550b

where:

• OD550e = Mean value of the measured optical density of the test item
• OD550b = Mean value of the measured optical density of the negative control

• Tetrazolium chloride
• Formazan

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• {{cite book | last = Wilson | first = Anne P. | editor-last = Masters | editor-first = John R. W. | name-list-style = vanc | year = 2000 | title = Animal Cell Culture: A Practical Approach | publisher = Oxford University Press | location = Oxford | edition = 3rd | volume = 1 | chapter = Chapter 7: Cytotoxicity and viability | isbn = 978-0-19-963796-6 | oclc = 43555390 | url = https://books.google.com/books?id=cU4120EgXZcC | lccn = 00026267 | access-date = 2016-09-24 | archive-date = 2020-04-14 | archive-url = https://web.archive.org/web/20200414144720/https://books.google.com/books?id=cU4120EgXZcC | url-status = live }}
• {{cite journal | vauthors = Bernas T, Dobrucki J | title = Mitochondrial and nonmitochondrial reduction of MTT: interaction of MTT with TMRE, JC-1, and NAO mitochondrial fluorescent probes | journal = Cytometry | volume = 47 | issue = 4 | pages = 236–242 | date = April 2002 | pmid = 11933013 | doi = 10.1002/cyto.10080 | doi-access = free }}

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• [https://www.scribd.com/doc/241231597/MTT-assay-Protocol MTT assay Protocol]

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Category:Biochemistry detection reactions