Dimethylthiazol-2-yl -2,5-diphenyl tetrazolium reduction

A tetrazolium salt (MTT) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide is able to enter cells and therefore assay levels of superoxide, without disrupting the cell with DMSO, forming the insoluble formazan product (Slater et al., 1963). Like the NBT test, MTT reduction is not specific for superoxide. 

Procedure Cell suspensions at 2 x 105/ml are maintained in Eagle s MEM supplemented with 10% (v/v) fetal calf serum. 

50 p of MTT solution (5 mg/ml in phosphate-buffered saline) are added to each well and the plates incubated at 37°C for the relevant time intervals. The medium containing MTT is then removed by aspiration very carefully and 100 p DMSO added to each well, and the plates gently agitated until the MTT formazan has dissolved. The absorbance of each well is read with a plate reader at 570 nm, subtracting the absorbance at 750 nm as a reference (there is no absorbance by MTT at 570 nm). The plates are read within 40 min of adding DMSO. 

There are disadvantages to the MTT assay The absorbance is preferably read within 1 h, as the absorbance decreases with time for example, 8% loss over 2 h after solubilization in DMSO. The major disadvantage is the limitation imposed by uncertainties in absorbance readings at low values. 

Advantages This assay in conjunction with the use of Multiwell plates, multichannel pipettes, microplate washer and shaker, together with a plate reading spectrophotometer, enables a large number of samples to be processed quickly. Interfacing the plate reader with a computer would provide an obvious additional advantage. 

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