Tetrazolium reduction

Early methods of superoxide detection are well known and described in many books and reviews. They include cytochrome c reduction, nitroblue tetrazolium reduction, spin trapping, etc. (see, for example, Ref. [1]). The most efficient assays are based on the ability of superoxide to reduce some compounds by one-electron transfer mechanism because such processes (Reaction (1)) proceed with high rates [2] ... 

Ries, W.L., Key, L.L., and Rodriguiz, R.M. 1992. Nitroblue tetrazolium reduction and bone resorption by osteoclasts in vitro inhibited by a manganese-based superoxide dismutase mimic. J. Bone Miner. Res. 7, 931-938. 

Among the first cell viability assays developed for HTS was the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] tetrazolium reduction assay (Mosmann 1983) that served as a milestone for this type of study. The assay offered a non-radioactive alternative to tritiated thymidine incorporation into DNA as a method of measuring cell proliferation. In many cases, the MTT assay can directly substitute for the tritiated thymidine incorporation assay (Figure 6.3). The MTT tetrazolium compound is prepared in a physiologically balanced solution, added to cells in culture, and incubated for approximately 4 hr. Viable cells convert MTT into an intensely colored formazan product that can be quantitated by recording changes in absorbance at specific wavelengths. 

Maly, F.-E. Nakamura, M. Gauchat, J.-F. Urwyler, A. Walker, C. Dahinden, C.A. Cross, A.R. Jones, O.T.G. Week, A.L. Superoxide-dependent nitroblue tetrazolium reduction and expression of cytochrome 6245 components by human tonsillar lymphocytes and B cell lines. J. Immunol. 1989, 142, 1260-1267. 

Nitro Blue Tetrazolium reduction under aerobic conditions is catalyzed by cetyltrimethyl ammonium bromide 92... 

Fe" " forms a colored complex with xylenol orange that can be read at 560 nm (Jones et al., 1995). Superoxide radical generation can be estimated by nitroblue-tetrazolium reduction assay (Libon et al., 1993). 

Stoddartet al. (1978) Lettuce hypocotyls membrane-based nonspecific tetrazolium reduction shows sudden rate change 14... 

Systems used to detect Oj include cytochrome c reduction (M4,M11,S4), nitroblue tetrazolium reduction (Bll,S3), adrenalin (M24,S19), hydroxyamine (M3), and a hydroxyamine derivative (E2,K14,K15). NADH/lactate dehydrogenase (B15), 6-hydroxydopamine (H10), sulfite (F8.G13), pyrogallol (M3), tetranitro-methane (M19), and dianisidine (G13.M24) are frequently employed. NAD(P)H oxidation has also been employed to generate Oj. In this system, NAD(P)H is the 0 detector (P2). SOD activity has also been estimated using hematoxylin autooxidation and chemiluminescence of a luciferin analog (K8,N6). In the case of illuminated flavin, nitroblue tetrazolium was used for the Oi detection (S3). 

Recently, the superoxide dismutase activity of low molecular mass copper chelates in the indirect coupled assay systems has been dispute It was demonstrated that copper in CuSO and Cu(II)(gly)2 prevents the ferricytochrome c and nitroblue tetrazolium reduction. This is not virtually new, as it is a well known phenomenon that Cu(II)-salts lead to a reoxidation of ferrocytochrome c and that they are potent inhibitors of xanthine oxidase which is often used as Oj" -generator in indirect SOD assay systems Although the indirect assays may be sometimes inadequate for the measurement of the SOD-activity, there are no doubts that low molecular mass copper chelates have their superoxide dismutase during pulse radiolysis. 

Fig. 1. DMSO induced differentiation of HL-60 cells. Nitro-blue-tetrazolium reduction ( ), phagocytosis of opsonised latex particles ( )...

In anaesthetised new-born pigs injury of moderate severity (1.9 to 2.1 atm) induced by the lateral fluid percussion brain injury technique staurosporine (10" M/1) blocked the nitroblue tetrazolium reduction after phorbol 12,13-dibutyrate and blunted the nitroblue tetrazolium reduction observed after lateral fluid percussion brain injury (1 1 to 15 2 versus 1 1 to 5 1 pmol/mm after lateral fluid percussion brain injury in the absence versus presence of staurosporine) (Armstead 1999). 

Naked monosodium urate crystals stimulate oxygen radical release by human polymorphonuclear leukocytes. Very recently, two papers dealing with superoxide anion generation by human neutrophils exposed to monosodium urate have been published. From the comparison of the data derived from nitroblue tetrazolium and cytochrome c reduction, it has been suggested that radical production in response to urate crystals is compartmentalized and occurs predominantly in the intracellular space. Microcrystalline sodium urate-induced oxygen consumption and nitroblue tetrazolium reduction were present for at least 15 min after stimulation of the cells. To enhance extracellular radicals neutrophils were converted to secretory non phagocitic cells by use of cytochalasin... 

Other forms of immunotoxic expression with occupational Pb exposures have been described. Brazilian Pb workers (Queiroz et al., 1993) had significantly reduced peripheral blood neutrophil populations versus controls, using both chemotaxis and nitroblue tetrazolium reduction. These impairments were seen in workers with PbB <60pg/dl. Mishra et al. (2003) examined immunotoxic effects of Pb in three groups of workers versus controls lead battery plant workers, jewelry makers, and three-wheeler drivers. Phytohemagglutinin-induced lymphocyte proliferation was inhibited versus controls, but no dose—response relationship was seen. T-cell mitogen-stimulated monocytes also showed an increase in IFN- but NK-cell level was unaffected. There was a positive correlation of IFN- with PbB. 

Phagocytosis, bactericidal activity and nitroblue tetrazolium reduction by the rabbit neutrophil in protein malnutrition, Indian J. Med. Res. 62 42. 

Cell viability Mitocbondrial enzymes activity MTT (3-(4,5-dimethylthiazol-2-yl) Tetrazolium reduction into formazan in metabolically active cells Optometric density... 

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