156:), 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 colormetric 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.)
162:(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. 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).
72:, which has a purple color. Other closely related tetrazolium dyes including XTT, MTS and the WSTs, are used in conjunction with the intermediate electron acceptor, 1-methoxy phenazine methosulfate (PMS). With WST-1, which is cell-impermeable, reduction occurs outside the cell via plasma membrane electron transport. However, this traditionally assumed explanation is currently contended as proof has also been found of MTT reduction to formazan in lipidic cellular structures without apparent involvement of oxidoreductases.
20:
171:
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. In addition, the mechanism of reduction of tetrazolium dyes,
179:
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
170:
Tetrazolium dye reduction is generally assumed to be dependent on NAD(P)H-dependent oxidoreductase enzymes largely in the cytosolic compartment of the cell. Therefore, reduction of MTT and other tetrazolium dyes depends on the cellular metabolic activity due to NAD(P)H flux. Cells with a low
75:
Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferation to quiescence) of potential medicinal agents and toxic materials. MTT assays are usually done in the dark since the MTT reagent is sensitive to light.
721:
Berridge MV, Tan AS (June 1993). "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".
135:(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.
129:. 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.
808:
Lyundup AV, Demchenko AG, Tenchurin TH, Krasheninnikov ME, Klabukov ID, Shepelev AD (2016). "Improving the seeding effectiveness of stromal and epithelial cell cultures in biodegradable matrixes by dynamic cultivation".
39:
for assessing cell metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium
404:
Stockert JC, Blázquez-Castro A, Cañete M, Horobin RW, Villanueva A (December 2012). "MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets".
462:"Comparative Study of in vitro Cytotoxicity of Povidone-Iodine in Solution, in Ointment or in a Liposomal Formulation (Repithel<sup>®</sup>) and Selected Antiseptics"
180:
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.
645:
Cory AH, Owen TC, Barltrop JA, Cory JG (July 1991). "Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture".
572:
Mosmann T (December 1983). "Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays".
138:
Water-soluble tetrazolium salts are more recent alternatives to MTT: they were developed by introducing positive or negative charges and
440:
183:
In studying the viability of cells seeded on 3D fibrous scaffolds, the thickness of the scaffolds may influence the MTT assay results.
152:(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), in the presence of phenazine methosulfate (
982:
927:"Mitochondrial and nonmitochondrial reduction of MTT: interaction of MTT with TMRE, JC-1, and NAO mitochondrial fluorescent probes"
617:
299:"Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives"
621:
356:
Berridge MV, Herst PM, Tan AS (2005). "Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction".
844:
517:"MTT versus other cell viability assays to evaluate the biocompatibility of root canal filling materials: a systematic review"
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195:(OD) at 550 nm is used to calculate the percentage of viability results using the following equation:
515:
Pintor, A. V. B.; Queiroz, L. D.; Barcelos, R.; Primo, L. S. G.; Maia, L. C.; Alves, G. G. (2020-07-27).
759:"The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis"
880:
106:
16:
Colorimetric analysis for measuring activity of cellular enzymes that reduce a tetrazolium dye
441:"Cytotoxicity Test by MTT Assay- Procedure, Analysis and Results - ACME Research Solutions"
220:
967:
8:
785:
758:
554:
497:
276:
99:
27:
after an MTT assay. Increasing amounts of cells resulted in increased purple colouring.
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24:
501:
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192:
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Wilson AP (2000). "Chapter 7: Cytotoxicity and viability". In
Masters JR (ed.).
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OD550b = Mean value of the measured optical density of the negative control
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48:
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247:"Cytotoxic Activity of Herbal Medicines as Assessed in Vitro: A Review"
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118:
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477:
693:
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Stockert JC, Horobin RW, Colombo LL, Blázquez-Castro A (April 2018).
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143:
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of this colored solution can be quantified by measuring at a certain
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88:
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OD550e = Mean value of the measured optical density of the test item
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114:
95:
69:
53:
65:
898:
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Ghasemi M, Turnbull T, Sebastian S, Kempson I (November 2021).
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92:
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Gavanji S, Bakhtari A, Famurewa AC, Othman EM (January 2023).
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19:
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to the phenyl ring of the tetrazolium salt, or better with
98:
in living cells. A solubilization solution (usually either
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groups added directly or indirectly to the phenyl ring.
102:, an acidified ethanol solution, or a solution of the
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113:) is added to dissolve the insoluble purple
763:International Journal of Molecular Sciences
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125:(usually between 500 and 600 nm) by a
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942:
876:Animal Cell Culture: A Practical Approach
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774:
532:
262:
684:"Water Soluble Tetrazolium Salts (WSTs)"
199:Viability % = 100 Ă— OD550e / OD550b
18:
724:Archives of Biochemistry and Biophysics
678:
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571:
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879:. Vol. 1 (3rd ed.). Oxford:
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438:
608:"Why should I use XTT instead of MTT"
117:product into a colored solution. The
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925:Bernas T, Dobrucki J (April 2002).
13:
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14:
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80:MTT and related tetrazolium salts
983:Biochemistry detection reactions
574:Journal of Immunological Methods
521:International Endodontic Journal
44:MTT, which is chemically 3-(4,5-
913:from the original on 2020-04-14
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703:from the original on 2018-12-22
627:from the original on 2018-04-06
460:MĂĽller, G.; Kramer, A. (2006).
439:Sharma, Dr Rudra (2022-10-05).
335:from the original on 2022-02-24
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1:
370:10.1016/S1387-2656(05)11004-7
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586:10.1016/0022-1759(83)90303-4
418:10.1016/j.acthis.2012.01.006
318:10.1016/j.acthis.2018.02.005
251:Chemistry & Biodiversity
7:
358:Biotechnology Annual Review
214:
10:
999:
659:10.3727/095535491820873191
175:intracellular (MTT, MTS)
881:Oxford University Press
823:10.5281/zenodo.1175840
736:10.1006/abbi.1993.1311
264:10.1002/cbdv.202201098
107:sodium dodecyl sulfate
28:
776:10.3390/ijms222312827
647:Cancer Communications
22:
221:Tetrazolium chloride
472:(Suppl. 1): 91–93.
68:, to its insoluble
968:MTT assay Protocol
944:10.1002/cyto.10080
845:"ISO 10993-5:2009"
100:dimethyl sulfoxide
37:colorimetric assay
29:
890:978-0-19-963796-6
534:10.1111/iej.13353
527:(10): 1348–1373.
478:10.1159/000090102
406:Acta Histochemica
306:Acta Histochemica
127:spectrophotometer
111:hydrochloric acid
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811:Genes to Cells
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58:-2-yl)-2,5-di
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915:. Retrieved
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166:Significance
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466:Dermatology
364:: 127–152.
257:(2): 3–27.
187:Observation
109:in diluted
87:, a yellow
63:tetrazolium
917:2016-09-24
854:2022-10-06
707:2013-08-12
631:2010-11-19
446:2022-10-06
339:2020-04-15
232:References
123:wavelength
119:absorbance
931:Cytometry
831:2313-1829
694:Interchim
559:220271896
543:0143-2885
486:1018-8665
281:255473013
144:sulfonate
104:detergent
89:tetrazole
33:MTT assay
977:Category
953:11933013
911:Archived
907:43555390
899:00026267
795:34884632
698:Archived
622:Archived
551:32602945
494:16490982
426:22341561
388:16216776
330:Archived
326:29496266
273:36595710
226:Formazan
215:See also
115:formazan
96:formazan
70:formazan
786:8657538
744:8390225
667:1867954
594:6606682
203:where:
66:bromide
55:thiazol
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93:purple
60:phenyl
50:methyl
701:(PDF)
625:(PDF)
555:S2CID
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333:(PDF)
302:(PDF)
277:S2CID
35:is a
949:PMID
903:OCLC
895:LCCN
885:ISBN
827:ISSN
791:PMID
740:PMID
663:PMID
590:PMID
547:PMID
539:ISSN
490:PMID
482:ISSN
422:PMID
384:PMID
374:ISBN
322:PMID
269:PMID
191:The
173:i.e.
160:WSTs
31:The
939:doi
849:ISO
819:doi
781:PMC
771:doi
732:doi
728:303
655:doi
582:doi
529:doi
474:doi
470:212
414:doi
410:114
366:doi
314:doi
310:120
259:doi
177:vs.
154:PMS
150:MTS
133:XTT
85:MTT
42:dye
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