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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 MTS tetrazolium compound is bioreduced by the mitochondrial activity of viable cells into a soluble colored formazan product, and the amount of colored formazan product is proportional to the number of viable cells.
After the formazan is formed, it is dissolved into a colored solution by adding a solubilizer such as sodium dodecyl sulfate.
Then, the 200 [micro]L of supernatant culture medium were carefully aspirated and 200 [micro]L aliquots of dimethylsulfoxide (DMSO) were added to each well to dissolve the formazan crystals after incubation for 10 min to dissolve air bubbles, the culture plate was placed on a Biotek micro-plate reader and the absorbance was measured at 550 nm.
After 24 hours, cell viability was assessed by pulsing the cells for 2 hours with dimethyl thiazolyl diphenyl tetrazolium salt (MTT) (5 mg/mL in PBS) followed by solubilization of Formazan crystals in 100 [micro]L of lysis buffer containing 20% sodium dodecyl sulfate and 50% dimethylformamide.
The yellow tetrazolium salt (MTT) is reduced by mitochondrial dehydrogenases in living cells to a blue-magenta coloured formazan precipitate [33].
In this assay live cells reduces the tetrazolium dye to its insoluble Formazan giving a purple coloure, whereas the dead cells does not show any reaction.
The reduced NADP in the presence of PMS caused the reduction of MTT to give a blue-coloured insoluble formazan, which was deposited at the sites of AK activity.
Supernatant was removed and the precipitated formazan was dissolved with 100 [micro]L dimethyl sulfoxide (Wolsen company, Xi'an, China) followed by gentle shaking of the dishes.
Mitochondrial succinate dehydrogenase in living cells converts MTT into visible formazan crystals during incubation.
In the NBT test, the blue dye formazan is produced by the reduction of the NBT dye, a reaction dependent upon neutrophil NADPH oxidase (2).
To avoid the interference created by ENMs while measuring formazan absorbance at 490 nm, the CPPs introduced a centrifugation (2000 x g for 10 min) procedure in phase II experiments to collect particles in the wells after incubation with the MTS reagents.
The viability of cells were assessed by the MTT (3-[4, 5 dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) assay, which is based on reduction of MTT by the mitochondrial dehydrogenase of intact cells to purple formazan products.