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A water-insoluble colored compound of the general structure, RNH-N=CR'-N=NR", formed by reduction of a tetrazolium salt in the histochemical demonstration of oxidative enzymes; the R's are usually phenyl groups; examples include neotetrazolium, blue tetrazolium, and nitro blue tetrazolium.
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The tetrazolium ring present in MTT is cleaved in active mitochondria, which results in production of purple colored formazan crystals.
The cells were washed and 150 uL Dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals.
The absorbance of formazan was measured using a microplate reader (Benchmark, Bio-Rad Laboratories, USA) at 570 nm.
Despues de 72 horas de incubacion en condiciones de microaerobiosis, se sustituye el medio por MTT (sal de tetrazolio), y se incuba durante 1 hora, produciendose la reduccion del MTT a formazan por las enzimas mitocondriales de las larvas viables y el cambio de color.
Formazan dissolved in organic solvent was added in each well and plates were placed on shaker for 30 min in dark.
The amount of formazan is directly proportional to the number of viable cells.
Afterwards, containing media were discarded and the formazan dye was solublized by adding 100 u L of DMSO (Fisher scientific CHEMTREC(r), USA) to each well.
When MTT reagent enters into the body, the live cells will produce an enzyme and break it to insoluble formazan crystals.
25) using tetrazolium MTT, 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (Sigma): seven-day culture after treatment for 72 h with the compounds were treated with a solution of MTT/PMS, and in viable cells mitochondria MTT salt was reduced to formazan by the action of succinate dehydrogenase; then a new treatment with HCl/SDS solution dissolves the formazan crystals.
In this method, MTT will be absorbed into mytochondria, then it will be reduced by reductase enzymes, and formazan crystals are formed.
Finally, 200 [micro]L of dimethyl sulfoxide was added to solubilize the formazan salt formed, and the amount of formazan salt was determined by measuring the OD at 540 nm using a GENios microplate reader (Tecan Austria GmbH, Grodig, Salzburg, Austria).
In this procedure, blood was mixed with nitroblue tetrazolium, allowed to incubate, smears were made, and counterstained with Leishman's stain and examined for the neutrophils containing formazan.