The MTT assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity. This colorimetric assay is based on the reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) to purple formazan crystals by metabolically active cells (Figure 1).6,7,35 The viable cells contain NAD(P)H-dependent oxidoreductase enzymes which reduce the MTT to formazan.36 The insoluble formazan crystals are dissolved using a solubilization solution and the resulting colored solution is quantified by measuring absorbance at 500-600 nanometers using a multi-well spectrophotometer. The darker the solution, the greater the number of viable, metabolically active cells.
This non-radioactive, colorimetric assay system using MTT was first described by Mosmann, T et al.1 and improved in subsequent years by several other investigators.2-6 The Cell Proliferation Kit I (MTT) is an optimized MTT assay kit containing ready to use reagents, does not need washing steps or additional reagents. It is a quantitative assay that allows rapid and convenient handling of a high number of samples. The Cell Proliferation Kit I (MTT) can be used for multiple applications, such as,
Figure 1.Metabolism of MTT to a formazan salt by viable cells as shown in a chemical reaction (A) and in a 96-well plate (B).
Figure 2.Determination of the cytotoxic activity of recombinant human TNF-α (hTNF-α) on WEHI-164 cells (mouse fibrosarcoma) using MTT assay.
Culture medium, e.g., DMEM (D5671) containing 10% heat inactivated FCS (fetal calf serum, 12106C), 2 mM glutamine (G6392), 0.55 mM L-arginine (A8094), 0.24 mM L-asparagine-monohydrate (A4284), 50 μM 2-mercaptoethanol (M3148), HT-media supplement (H0137) (1×), containing 0.1 mM hypoxanthine and 16 μM thymidine. If an antibiotic is to be used, additionally supplement media with penicillin/streptomycin or gentamicin. Interleukin-6, human (hIL-6, SRP3096) (200,000 U/ml, 2 μg/ml) sterile.
For the determination of human interleukin-6 (hIL-6) activity on 7TD1 cells (mouse-mouse hybridoma, DSMZ, ACC 23) (see fig. 3).
Figure 3.Proliferation of 7TD1 cells (mouse-mouse hybridoma) in response to recombinant human interleukin-6 (hIL-6) using MTT assay.
XTT assay and WST-1 assay can also be used for measuring cell viability and proliferation.