Formazan

Formazan dyes are artificial chromogenic products of the reduction of tetrazolium salts by dehydrogenases and reductases. They have a variety of colors from dark blue to deep red to orange, depending on the original tetrazolium salt used as the substrate for the reaction.

Reduction of a tetrazolium to a formazan. The "Rs" stand-in for various organic groups that define the various tetrazolium salts and provide their unique chemical characteristics.
MTT test: Formation of formazan crystals from MTT in mesenchymal stem cells

Leading examples of tetrazolium salts include:[1]

  1. INT or 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride, which is water-insoluble.
  2. MTT or 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide, which is water-insoluble and used in the MTT assay.
  3. XTT or 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, which is water-soluble.
  4. MTS or 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, which is water-soluble and used in the MTS assay.
  5. TTC or tetrazolium chloride or 2,3,5-triphenyl-2H-tetrazolium chloride, which is water-soluble.
  6. NBT used in a diagnostic test, particularly for chronic granulomatous disease and other diseases of phagocyte function.

When reduced in a cell, either enzymatically or through direct reaction with NADH or NADPH, the classical tetrazolium salt, MTT, turns blue to purple and may form an insoluble precipitate.[2][3] These formazan dyes are commonly used in cell proliferation and toxicity assays such as the EpiDerm[4] and EpiSkin tests since they only stain living, metabolically active cells.[5][6]

References
  1. Altman FP (1976). "Tetrazolium salts and formazans". Prog. Histochem. Cytochem. 9 (3): 1–56. doi:10.1016/S0079-6336(76)80015-0. PMID 792958.
  2. Stockert, Juan C.; Horobin, Richard W.; Colombo, Lucas L.; Blázquez-Castro, Alfonso (2018). "Tetrazolium salts and formazan products in Cell Biology: Viability assessment, fluorescence imaging, and labeling perspectives" (PDF). Acta Histochemica. 120 (3): 159–167. doi:10.1016/j.acthis.2018.02.005. PMID 29496266.
  3. Stockert, Juan C.; Blázquez-Castro, Alfonso; Cañete, Magdalena; Horobin, Richard W.; Villanueva, Ángeles (2012). "MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets". Acta Histochemica. 114 (8): 785–796. doi:10.1016/j.acthis.2012.01.006. PMID 22341561.
  4. Mattek
  5. Marshall NJ, Goodwin CJ, Holt SJ (June 1995). "A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function". Growth Regul. 5 (2): 69–84. PMID 7627094.
  6. Scudiero DA, Shoemaker RH, Paull KD, et al. (1 September 1988). "Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines". Cancer Res. 48 (17): 4827–33. PMID 3409223.


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