Briefly, sections of fresh tissue from the lumbosacral spinal cord were mixed with NBT to form formazan
as an index of superoxide anion generation.
Cell viability was assayed by measuring blue formazan
that was metabolized from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by mitochondrial dehydrogenase.
We also pipetting the liquid m to fully dissolve the MTT formazan
. We read absorbance at 0D=590 nm within 15 minutes.
Normal neutrophils reduce nitroblue-tetrazolium to formazan
, a dark blue pigment visible on microscopic inspection.
The assay assesses cell viability by detecting the formazan
product formed from the reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase of metabolically active cells .
After 96 h, MTT solution (20 uL, 5 mg/mL in PBS) was added in each well and the plate was then again incubated for 4 h for MTT formazan
The principle of the assay is based on generation of O2-*, which is released during xanthine oxidation and xanthine oxidase, to form the formazan
by reducing nitroblue tetrazolium (NBT).
The quantity of formazan
(presumably directly proportional to the number of viable cells) was measured by recording changes in absorbance at 570 nm using a plate reading spectrophotometer.
2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay is based on the reduction of XTT tetrazolium formazan
by active mitochondria in Candida cells (28).
The WST-1 assay used to determine cell proliferation is based on the basic principle the conversion of the pink tetrazolium salt (WST-1) to a dark red formazan
dye via the succinate-tetrazolium reductase enzyme, which is active only in living cells in the mitochondrial respiratory chain .
It involves generation of superoxide by pyrogallol autoxidation and inhibition of superoxide dependent reduction of tetrazolium dye MTT to its formazan
. The reaction was terminated, formazan
This method depends on the cleavage of the stable tetrazolium salt to soluble formazan
. The amount of formazan
produced indicates the metabolically active cells.