Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability.
A convenient way to estimate the number of viable cells growing in microtitre tray wells is to use a colorimetric assay and an automatic microplate scanning spectrophotometer. One such assay, developed by Mosmann, depends on the reduction by living cells of tetrazolium salt, MTT, to form a blue formazan product. However the original technique has several technical limitations, namely a less than optimal sensitivity, a variable background due to protein precipitation on adding an organic solvent to dissolve the blue formazan product, and a low solubility of the product. These problems have been overcome by the following modifications: avoidance of serum in the incubation medium, thus overcoming precipitation problems in the organic solvent; avoidance of phenol red in the incubation medium, thus avoiding the use of acid in the final solvent which altered the spectral properties of the formazan; elimination of the medium containing MTT after the reaction and subsequent use of pure propanol or ethanol to rapidly solubilize the formazan; use of a higher concentration of MTT; use of half-area microtitre trays to increase the spectrophotometer readings from a given amount of formazan; use of a more judicious reference wavelength in a dual wavelength spectrophotometer. With these modifications the reliability and sensitivity of the test have been increased to the point where it can in many cases replace the [3H]thymidine uptake assay to measure cell proliferation or survival in growth factor or cytotoxicity assays. Examples of its use in IL-2 assays are given.
D000818:Animals; D002455:Cell Division; D002460:Cell Line; D002470:Cell Survival; D003124:Colorimetry / Q000379:methods* / Q000592:standards; D003470:Culture Media; D007376:Interleukin-2 / Q000032:analysis*; D008213:Lymphocyte Activation; D051379:Mice; D010637:Phenolsulfonphthalein; D013778:Tetrazolium Salts; D013936:Thymidine / Q000378:metabolism
D003470:Culture Media; D007376:Interleukin-2; D013778:Tetrazolium Salts; D010637:Phenolsulfonphthalein; D013936:Thymidine