MTT assay
The MTT assay and the MTS assay are colorimetric assays for measuring the activity of enzymes that reduce MTT or close dyes (XTT, MTS, WSTs) to formazan dyes, giving a purple color. A main application allows to assess the viability (cell counting) and the proliferation of cells (cell culture assays). It can also be used to determine cytotoxicity of potential medicinal agents and toxic materials, since those agents would stimulate or inhibit cell viability and growth.
MTT and related tetrazolium salts
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole), is reduced to purple formazan in living cells.[1] 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 absorption maximum is dependent on the solvent employed.
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 dyes is water soluble, avoiding a final solubilization step[2].
Water soluble tetrasolium 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.
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 490-500 nm in phosphate-buffered saline.[3]
WSTs (Water soluble Tetrazolium salts) are a series of other water soluble dyes for MTT Assays, developped to give different absorption spectra of the formed formazans[4]. 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, WTS assays (1) can be read directly (unlike MTT that needs a solubilization step), (2) give a more effective signal than MTT, and (2) decrease toxicity to cells (unlike cell-permeable MTT, and its insoluble formazan that accumulate inside cells)[4].
MTT Assays significance
These reductions take place only when reductase enzymes are active, and therefore conversion is often used as a measure of viable (living) cells. However, it is important to keep in mind that other viability tests (such as the CASY cell counting technology) sometimes give completely different results, as many different conditions can increase or decrease metabolic activity. Changes in metabolic activity can give large changes in MTT or MTS results while the number of viable cells is constant. When the amount of purple formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control cells, the effectiveness of the agent in causing death, or changing metabolism of cells, can be deduced through the production of a dose-response curve.
References
- ^ 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. PMID 6606682.
{{cite journal}}: Unknown parameter|month=ignored (help) - ^ Comparison of XTT versus MTT by Aniara.pdf
- ^ Cory AH, Owen TC, Barltrop JA, Cory JG (1991). "Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture". Cancer communications. 3 (7): 207–12. PMID 1867954.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ a b water soluble tetrazolium salts WST-1 to WST-11 technical manual
- Wilson, A. P., Cytotoxicity and Viability Assays in Animal Cell Culture: A Practical Approach, 3rd ed. (ed. Masters, J. R. W.) Oxford University Press: oXford 2000, Vol. 1,
- Mitochondrial and nonmitochondrial reduction of MTT: interaction of MTT with TMRE, JC-1, and NAO mitochondrial fluorescent probes. Bernas T, Dobrucki J. Cytometry. 2002 Apr 1;47(4):236-42. PMID 11933013