cell viability/proliferation kit (MTT-assay, XTT assay WST-1)

Tetrazolium salts (e.g., MTT, XTT, WST-1) are particularly useful for cell viability & proliferation. They are cleaved to formazan by the 'succinate-tetrazolium reductase' system (EC 1.3.99.1), a component of the mitochondrial respiratory chain. This it is only active in metabolically intact cells. MTT assay Assay uses: 1) quantitative determination of cellular proliferation & activation - response to growth factors or cytokines) 2) quantitation of antiproliferative or cytotoxic effects 3) quantitation of in vitro tumor cell chemosensitivity 4) assessment of photoradiation therapy 5) screening of anticancer compounds 6) MTT dissolved in agarose overlays is used to improve visualization of virus-infected cells in plaque assays Advantages: - no radioactive isotopes are used - tetrazolium-salt conversion strongly correlates with metabolic acvtivity - low cell numbers can be used - the use of multi-well ELISA readers allows processing of large numbers of samples - no washing steps or additional reagents are required Principle: 1) based on the cleavage of the yellow tetrazolium salt MTT into purple formazan by metabolically active cells 2) MTT formazan crystals are insoluble in aqueous solution, but may be solubilized (by addition of solution included in the kit & incubating overnight 37 C, 6.5% CO₂) 3) solubilized formazan product can be photometrically quantitated using an ELISA reader 4) an increase in the number of living cells results in an increase of total metabolic activity which leads to more color formation XTT assay Assay uses: - used for the same applications as the MTT assay Assay principle: 1) based on the conversion of the yellow tetrazolium salt XTT into an orange formazan dye by metabolically active cells 2) in contrast to the purple formazan crystals in the MTT assay, the XTT formazan dye is soluble in aqueous solutions, thus be directly quantitated with an ELISA reader WST-1 assay Assay principle: 1) WST-1 yields water-soluble cleavage products, like XTT, which can be measured directly 2) see XTT assay WST-1 advantages over XTT: 1) WST-1 is more stable than XTT 2) WST-1 can be used as a ready-to-use solution & can be stored at 4 C for several weeks without significant degradation 3) WST-1 has a wider linear range & shows accelerated color development compared to XTT

References

Roche Diagnostics, Apoptosis and Cell Proferation http://www.roche-applied-science.com/fst/apoptosis.htm?/sis/apoptosis/products/cell_proliferation_kits.htm