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PubMed1998

Subcellular localization of the neuronal isoform of nitric oxide synthase in the rat brain: a critical evaluation.

In the aldehyde-fixed rat brain NADPH-diaphorase is suggested to be related to brain nitric oxide synthase but also to other isoforms of this enzyme as well as to several non-related types of NADPH-oxidoreductases. In this study NADPH-diaphorase histochemistry using the tetrazolium salt BSPT (2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl)-tetrazoliu m chloride) (to yield an electron dense formazan) and immunocytochemistry were applied for the cellular and subcellular localization of brain nitric oxide synthase in the striatum and the pontine laterodorsal tegmental nucleus of the rat. Combining the two techniques, in both brain regions identical distribution patterns of heavily-stained neurons were observed at the light microscopic level. There are inconsistencies in the literature with regard to the subcellular localization of brain nitric oxide synthase and NADPH-diaphorase in neurons. In our results brain nitric oxide synthase immunoreactivity in abundantly stained neurons was mainly cytosolically distributed, sometimes in a patch-like form and distant from membranes, whereas the NADPH-diaphorase reaction product BSPT-formazan was closely attached to discrete portions of intracellular membranes. Other neurons and glial cells including their processes showed also, but to a lesser extent, formazan-labelled membrane portions. In such cell populations brain nitric oxide synthase immunoreactivity was not detectable. Possible reasons for these inconsistencies are discussed in detail. The strength but not the specificity of the NADPH-diaphorase related reaction was shown to be dependent on concentrations of Triton X-100 and tetrazolium salt. We suggest that, for electron microscopical cytochemistry, the BSPT technique combined with other independent techniques, such as immunocytochemistry and in situ hybridization, may be a viable means for the identification and subcellular localization of the different nitric oxide synthase isoforms, and to discriminate them from other types of NADPH-diaphorases.

Animals↗

PubMed1990

A feasibility study of the MTT assay for chemosensitivity testing in ovarian malignancy.

We assess the feasibility of using the MTT assay as a measure of cell viability in chemosensitivity testing in ovarian malignancy. The assay utilises the conversion of the tetrazolium salt MTT to formazan by dehydrogenase enzymes in living cells. We show that the optical density of the formazan produced from MTT is directly proportional to the number of live cells tested. Optimum MTT conversion occurred after 4 h incubation and dimethyl sulphoxide was found to be the most suitable solvent for the formazan. Seventy-five samples of ascitic fluid and/or solid tumour were collected from 56 patients with FIGO stage III-IV ovarian adenocarcinoma. Malignant cell suspensions with a viability greater than 75% were prepared from 95% of ascitic fluid and 75% of biopsy samples by simple techniques. The effect of cytotoxic drugs was assessed in 91% of patients included in the study. Variation in drug effect between patients was evident following a 48 h incubation period and was reproducible. Overall platinum and anthraquinone analogues produced the greater effect but resistance did occur. Our results mirrored reported clinical response rates. Only one sample tested against chlorambucil showed any drug effect. As this assay produces results in a high percentage of tests and is rapid and simple it appears suitable for prospective clinical trials to correlate the in vitro results with in vivo response.

Adenocarcinoma↗

PubMed1975

The nylon column dye test: a possible screening test of phagocyte function.

1. A simple quantitative test has been developed to investigate phagocyte function. 2. This test is performed by the addition of Nitroblue Tetrazolium to whole blood, followed by the isolation of leucocytes on a column of nylon wool. Dye reduction by phagocytes is apparent as a blue coloration of the column due to the formation of formazan. The formazan can be extracted from the column and measured spectrophotometrically. 3. The formation of formazan was found to be directly related to the number of phagocytes in blood. 4. Two patients with chronic granulomatous disease gave abnormal results, suggesting that the test procedure may be of value as a screening procedure for this disease.

Adult↗

PubMedDate not supplied

Cholesterol interferes with the MTT assay in human epithelial-like (A549) and endothelial (HLMVE and HCAE) cells.

Metabolically active cells are able to convert the MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye to blue formazan. This is the basis of the MTT assay, which is among the most widely used screening methods to evaluate cell viability and proliferation. When testing the effects of cholesterol products on the viability of human pulmonary epithelial-like A549 cells using trypan blue staining (cell numbers) and the MTT assay, results were inconsistent. The MTT assay indicated greater than 50% loss of viability with exposure of cells to cholesterol, whereas there was no decrease in viability indicated by trypan blue exclusion and propidium iodide uptake. A similar decrease in MTT reduction was obtained upon cholesterol treatment in human lung microvascular endothelial cells (HLMVECs) and human coronary artery endothelial cells (HCAECs) without loss of viability. This suggested a direct interference of cholesterol with the assay. However, using a cell-free system, there was no decrease in the reduction of MTT by ascorbic acid during incubation with a similar concentration of cholesterol. Light microscopy revealed enhanced exocytosis of formazan granules in presence of cholesterol. Incubation with apolipoprotein A-1 decreased cholesterol-mediated inhibition of MTT assay. These studies indicate decreased MTT reduction as a result of enhanced exocytosis of formazan due to cholesterol. A careful validation of viability assay procedures is therefore suggested in experiments where cholesterol is a constituent, to avoid a potential bias in concluding results of cytotoxicity studies.

Apolipoprotein A-I↗

PubMed1963

The use of tetranitro-blue tetrazolium for the cytochemical localization of succinic dehydrogenase. Cytochemical and cytological studies of sarcoma 37 ascites tumor cells.

Succinic dehydrogenase (SDH) was localized in the mitochondria of Sarcoma 37 ascites tumor cells by the use of tetranitro-BT (TNBT) and nitro-BT (NBT) in smear preparations. Results with each tetrazolium salt as electron acceptor were evaluated with respect to: (a) size and shape of the formazan precipitate relative to standard mitochondrial morphology; (b) crystallization phenomena of reduced dye; (c) lipid adsorption of formazan. The association of formazan- or iron hematoxylin-stained mitochondria with lipid droplets within the cells was investigated, as was also the influence of formalin fixation, with and without cold acetone pretreatment, on mitochondrial morphology and enzymatic staining. Data from these studies appear to indicate that TNBT is more suitable than NBT for use as a cytochemical reagent in oxidative and/or dehydrogenase enzyme histochemistry and cytochemistry.

Animals↗

PubMed1958

The cytochemical localization of oxidative enzymes. I. Diphosphopyridine nucleotide diaphorase and triphosphopyridine nucleotide diaphorase.

Cytochemical methods involving metal chelation of the formazan of an N-thiazol-2-yl tetrazolium salt are described for the localization of diphosphopyridine nucleotide diaphorase (DPND) and triphosphopyridine nucleotide diaphorase (TPND) in mitochondria. These methods utilize the reduced coenzymes DPNH or TPNH as substrate. The reaction involves a direct transfer of electrons from reduced coenzyme to the respective diaphorase which in turn transfers the electrons to tetrazolium salt, reducing it to the insoluble formazan. Competition for electrons by preferential acceptors in the respiratory chain was prevented by various inhibitors. In the presence of respiratory inhibitors the rate of tetrazolium reduction was markedly increased. The greatest reduction was observed when amytal was used. Sites of diaphorase activity appeared as deposits of blue-black metal formazan chelate measuring 0.2 to 0.3 micro in diameter. Small mitochondria contained 2 deposits, while larger ones contained up to 6. Considerable differences were observed in the rate of tetrazolium reduction and cellular localization of diaphorase activity when DPNH was used as substrate as compared to TPNH. In each instance DPNH was oxidized more rapidly by tissues than TPNH. These findings support the concept that the oxidation of coenzymes I and II is mediated through separate diaphorases.

Coenzymes↗

PubMed1961

Mitochondrial localization of oxidative enzymes: staining results with two tetrazolium salts.

A comparison is made of the staining results obtained with Nitro-BT and MTT-Co(++) as acceptors when DPNH is the substrate in frozen sections of cold formol-calcium-fixed rat kidney (normal and following ligation of the blood vessels) and human liver containing lipofuscin granules. The kidney results are evaluated in terms of mitochondrial morphology seen after classical mitochondrial stains and in electron micrographs. It is concluded that, except for formazan deposition on lipid-aqueous interfaces, Nitro-BT staining indicates the intracellular localization of oxidative enzymes, at least at the level of light microscopy. In contrast, the use of MTT-Co(++) is not reliable for such intracellular localizations. The deposition of the formazan of MTT-Co(++) is determined in large part by physicochemical factors other than enzyme localization. Despite marked abnormalities of the mitochondria in cells of the ligated kidney, MTT-Co(++) formazan is generally deposited in the same dotlike fashion as in cells of normal kidney.

Animals↗

PubMed1989

Arachidonic acid potentiates superoxide anion radical production by murine peritoneal macrophages stimulated with tumor promoters.

The role of arachidonic acid (AA) metabolism in the stimulation of oxygen radical production by murine peritoneal macrophages treated with tumor promoters was assessed. In vivo administration of the phospholipase A2 inhibitor dibromacetophenone, the anti-inflammatory steroid fluocinolone acetonide or the lipoxygenase inhibitor nordihydroguiaretic acid just prior to i.p. injection of phorbol-12-myristate-13-acetate (PMA, 100 ng) into unmanipulated CD-1 female mice resulted in a dose-dependent decrease in the number of peritoneal exudate cells (PEC) producing superoxide anion radical (O2) as assessed by the reduction of nitroblue tetrazolium, i.e. the formation of formazan-positive PEC. The cycloxygenase inhibitor indomethacin had no effect on the number of formazan-positive PEC caused by PMA treatment. The ability of PMA, phorbol-12,13-dibutyrate mezerein, phorbol-12,13-diacetate and 4-O-Me-PMA to stimulate the production of oxygen radicals by murine peritoneal macrophages correlated with their ability to stimulate the release of [3H]AA equivalents from the macrophages. The calcium ionophore A23187 which stimulated significant [3H]AA equivalent release did not stimulate superoxide anion radical production by the macrophages. PMA administered i.p. to SENCAR mice increased the number of formazan-positive PEC 4-to 5-fold compared with similarly treated C57BL/6 mice. PMA also stimulated the release of twice the amount of [3H]AA equivalents from peritoneal macrophages from SENCAR mice compared with that released by macrophages from C57BL/6 mice. The addition of low concentrations of AA (1-10 microM) in vitro to casein-elicited murine peritoneal macrophages treated with low concentrations of PMA (1 ng/ml) resulted in a 2-fold potentiation of the amount of superoxide anion radical produced compared with PMA treatment alone as assessed by the reduction of cytochrome c. These results demonstrate that AA and/or a lipoxygenase product can potentiate the production of oxygen radicals by murine peritoneal macrophages treated with tumor promoters.

Animals↗

PubMed1989

Tumor promoters differ in their ability to stimulate superoxide anion radical production by murine peritoneal exudate cells following in vivo administration.

The production of superoxide anion radicals by murine peritoneal exudate cells (PECs) following intraperitoneal (i.p.) injection of mouse skin tumor promoters was assessed in vitro by the reduction of nitroblue tetrazolium (NBT). Phorbol-12-myristate-13-acetate (PMA) or mezerein when administered i.p. to CD-1 female mice at a dose of 0.1 microgram caused an inflammatory response in the peritoneal cavity. PMA (0.1 microgram) also caused an increase in the number of PEC reducing NBT, i.e. formazan-positive PECs (20% positive cells) as compared to vehicle control from 15 min through 2 h following i.p. administration to unmanipulated mice. In the same system, dose-response studies demonstrated that 4-O-methyl-phorbol myristate acetate (4-O-Me-PMA) and mezerein were approximately 50 times less active than PMA, while phorbol dibutyrate (PDB), phorbol diacetate (PDA), phorbol and the calcium ionophore A23187 did not cause an increase in the number of formazan-positive PECs at the doses used. When the same compounds (0.1 microgram) were administered to mice which had been pretreated i.p. with thioglycollate 6 days prior to the experiment, PMA, PDB, 4-O-Me-PMA, mezerein and A23187 all caused an increase in the number of formazan-positive PECs 2 h after treatment. The PEC which reduced NBT in response to tumor promoters were predominantly adherent and esterase positive cells, suggesting they were macrophages. These results indicate that in vivo administration of tumor promoters results in the production of superoxide anion radicals by murine peritoneal macrophages and that the physiological state of the macrophages is important in the response to tumor promoters.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

A simplified new assay for assessment of fungal cell damage with the tetrazolium dye, (2,3)-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanil ide (XTT).

Studies of antimycotic host defenses have been limited by the paucity of rapid, reproducible quantitative assays for fungal cell damage. Prior studies defined a colorimetric method that uses MTT, a tetrazolium dye, to quantify polymorphonuclear leukocyte (PMNL)-mediated damage to fungi. These relatively simple, rapid, and reproducible assays require cumbersome extraction of precipitated MTT-formazan and high cell densities to overcome relatively low sensitivity. In experiments that compared assays with MTT and another tetrazolium dye, 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-+ ++carboxanilide (XTT), estimates of damage to Aspergillus fumigatus hyphae by human PMNL were similar with both dyes. However, XTT reduction was more rapid and sensitive, yielding accurate results with fewer organisms and PMNL. The water-soluble XTT-formazan product also simplified measurements by eliminating the need for solvent-extraction steps that are obligatory in MTT assays. Thus, XTT is advantageous for quantitative assessment of fungal cell damage, although MTT remains useful for assessing fungal cell viability by direct microscopic visualization of precipitated formazan.

Aspergillus fumigatus↗

PubMed2001

Superoxide production by plant homologues of the gp91(phox) NADPH oxidase. Modulation of activity by calcium and by tobacco mosaic virus infection.

Genes encoding homologs of the gp91(phox) subunit of the plasma membrane NADPH oxidase complex have been identified in plants and are hypothesized to be a source of reactive oxygen species during defense responses. However, the direct involvement of the gene products in superoxide (O(2)(-)) production has yet to be shown. A novel activity gel assay based on protein fractionation in native or sodium dodecyl sulfate (SDS)-denaturing polyacrylamide gels was developed. In native polyacrylamide gel electrophoresis, one or two major O(2)(-)-producing formazan bands were detected in tomato (Lycopersicum esculentum Mill. cv Moneymaker) and tobacco (Nicotiana tabacum var. Samsun, NN) plasma membranes, respectively. Denaturing fractionation of tomato and tobacco plasma membrane in SDS-polyacrylamide gel electrophoresis, followed by regeneration of the in-gel activity, revealed NADPH-dependent O(2)(-)-producing formazan bands of 106-, 103-, and 80- to 75-kD molecular masses. The SDS and native activity bands were dependent on NADPH and completely inhibited by diphenylene iodonium or CuZn- O(2)(-) dismutase, indicating that the formazan precipitates were due to reduction by O(2)(-) radicals catalyzed by an NADPH-dependent flavin containing enzyme. The source of the plasma membrane activity bands was confirmed by their cross-reaction with antibody prepared from the C terminus of the tomato gp91(phox) homolog. Membrane extracts as well as the in-gel NADPH oxidase activities were stimulated in the presence of Ca(2+). In addition, the relative activity of the gp91(phox) homolog was enhanced in the plasma membrane of tobacco mosaic virus-infected leaves. Thus, in contrast to the mammalian gp91(phox), the plant homolog can produce O(2)(-) in the absence of additional cytosolic components and is stimulated directly by Ca(2+).

Calcium↗

PubMed1982

Improved method for determination of respiring individual microorganisms in natural waters.

A method is reported that combines the microscopic determinations of specific, individual, respiring microorganisms by the detection of electron transport system activity and the total number of organisms of an estuarine population by epifluorescence microscopy. An active cellular electron transport system specifically reduces 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan, which is recognized as opaque intracellular deposits in microorganisms stained with acridine orange. In a comparison of previously described sample preparation techniques, a loss of >70% of the counts of INT-reducing microorganisms was shown to be due to the dissolution of INT-formazan deposits by immersion oil (used in microscopy). In addition, significantly fewer fluorescing microorganisms and INT-formazan deposits, both </=0.2 mum in size, were found for sample preparations that included a Nuclepore filter. Visual clarity was enhanced, and significantly greater direct counts and counts of INT-reducing microorganisms were recognized by transferring microorganisms from a filter to a gelatin film on a cover glass, followed by coating the sample with additional gelatin to produce a transparent matrix. With this method, the number of INT-reducing microorganisms determined for a Chesapeake Bay water sample was 2-to 10-fold greater than the number of respiring organisms reported previously for marine or freshwater samples. INT-reducing microorganisms constituted 61% of the total direct counts determined for a Chesapeake Bay water sample. This is the highest percentage of metabolically active microorganisms of any aquatic population reported using a method which determines both total counts and specific activity.

Journal Article↗

PubMed1998

Protein method for investigating mercuric reductase gene expression in aquatic environments.

A colorimetric assay for NADPH-dependent, mercuric ion-specific oxidoreductase activity was developed to facilitate the investigation of mercuric reductase gene expression in polluted aquatic ecosystems. Protein molecules extracted directly from unseeded freshwater and samples seeded with Pseudomonas aeruginosa PU21 (Rip64) were quantitatively assayed for mercuric reductase activity in microtiter plates by stoichiometric coupling of mercuric ion reduction to a colorimetric redox chain through NADPH oxidation. Residual NADPH was determined by titration with phenazine methosulfate-catalyzed reduction of methyl thiazolyl tetrazolium to produce visible formazan. Spectrophotometric determination of formazan concentration showed a positive correlation with the amount of NADPH remaining in the reaction mixture (r2 = 0.99). Mercuric reductase activity in the protein extracts was inversely related to the amount of NADPH remaining and to the amount of formazan produced. A qualitative nitrocellulose membrane-based version of the method was also developed, where regions of mercuric reductase activity remained colorless against a stained-membrane background. The assay detected induced mercuric reductase activity from 10(2) CFU, and up to threefold signal intensity was detected in seeded freshwater samples amended with mercury compared to that in mercury-free samples. The efficiency of extraction of bacterial proteins from the freshwater samples was (97 +/- 2)% over the range of population densities investigated (10(2) to 10(8) CFU/ml). The method was validated by detection of enzyme activity in protein extracts of water samples from a polluted site harboring naturally occurring mercury-resistant bacteria. The new method is proposed as a supplement to the repertoire of molecular techniques available for assessing specific gene expression in heterogeneous microbial communities impacted by mercury pollution.

Bacteria↗

PubMed1980

Assay of succinate dehydrogenase activity by the tetrazolium method: evaluation of an improved technique in skeletal muscle fractions.

An improved spectrophotometric method for measuring succinate dehydrogenase (EC 1.3.99.1) activity with the use of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) is described. The procedure has been evaluated in mitochondrial fractions and homogenates of frog skeletal muscle. For mitochondrial suspensions, extraction of formazan with alcohol was found to be superior to extraction with ethyl acetate. For homogenates, complete extraction of formazan required sequential treatment with alcohol and ethyl acetate; the generally employed procedure of extracting once with ethyl acetate alone led to serious underestimation of the amount of formazan in the tissue. Observations of mitochondrial suspension incubated with various concentrations of INT led to the selection of 0.8 mM INT for optimal results. Higher concentrations, although commonly used, can exert undesirable inhibitory effects on succinate dehydrogenase activity, especially at low concentrations of mitochondria and after longer periods of incubation. The problem of instability of succinate dehydrogenase was solved by the addition of buffer at pH 7.5.

Animals↗

PubMed2006

Onchocerca parasites and Wolbachia endosymbionts: evaluation of a spectrum of antibiotic types for activity against Onchocerca gutturosa in vitro.

BACKGROUND: The filarial parasites of major importance in humans contain the symbiotic bacterium Wolbachia and recent studies have shown that targeting of these bacteria with antibiotics results in a reduction in worm viability, development, embryogenesis, and survival. Doxycycline has been effective in human trials, but there is a need to develop drugs that can be given for shorter periods and to pregnant women and children. The World Health Organisation-approved assay to screen for anti-filarial activity in vitro uses male Onchocerca gutturosa, with effects being determined by worm motility and viability as measured by reduction of MTT to MTT formazan. Here we have used this system to screen antibiotics for anti-filarial activity. In addition we have determined the contribution of Wolbachia depletion to the MTT reduction assay. METHODS: Adult male O. gutturosa were cultured on a monkey kidney cell (LLCMK 2) feeder layer in 24-well plates with antibiotics and antibiotic combinations (6 to 10 worms per group). The macrofilaricide CGP 6140 (Amocarzine) was used as a positive control. Worm viability was assessed by two methods, (i) motility levels and (ii) MTT/formazan colorimetry. Worm motility was scored on a scale of 0 (immotile) to 10 (maximum) every 5 days up to 40 days. On day 40 worm viability was evaluated by MTT/formazan colorimetry, and results were expressed as a mean percentage reduction compared with untreated control values at day 40. To determine the contribution of Wolbachia to the MTT assay, the MTT formazan formation of an insect cell-line (C6/36) with or without insect Wolbachia infection and treated or untreated with tetracycline was compared. RESULTS: Antibiotics with known anti-Wolbachia activity were efficacious in this system. Rifampicin (5 x 10(-5) M) was the most effective anti-mycobacterial agent; clofazimine (1.25 x 10(-5) M and 3.13 x 10(-6) M) produced a gradual reduction in motility and by 40 days had reduced worm viability. The other anti-mycobacterial drugs tested had limited or no activity. Doxycycline (5 x 10(-5) M) was filaricidal, but minocycline was more effective and at a lower concentration (5 x 10(-5) M and 1.25 x 10(-5) M). Inactive compounds included erythromycin, oxytetracycline, trimethoprim and sulphamethoxazole. The MTT assay on the insect cell-line showed that Wolbachia made a significant contribution to the metabolic activity within the cells, which could be reduced when they were exposed to tetracycline. CONCLUSION: The O. gutturosa adult male screen for anti-filarial drug activity is also valid for the screening of antibiotics for anti-Wolbachia activity. In agreement with previous findings, rifampicin and doxycycline were effective; however, the most active antibiotic was minocycline. Wolbachia contributed to the formation of MTT formazan in the MTT assay of viability and is therefore not exclusively a measure of worm viability and indicates that Wolbachia contributes directly to the metabolic activity of the nematode.

Journal Article↗

PubMed2004

Inaccuracies in MTS assays: major distorting effects of medium, serum albumin, and fatty acids.

Soluble formazan assays are widely used for cell number assessment. However, in our hands, we observed frequent occasions in which the actual cell number was at odds with the assay reading. In this study, we have determined that (i) a large proportion of the reading obtained in commonly used culture media can be caused by media component amplification of formazan production in a way that cannot be corrected for by media-only controls; (ii) the albumin present in 10% serum can reduce the assay absorbance by 50% so that an actual doubling of cell number can be obscured; and (iii) this latter effect is dependent on the concentration of fatty acids. To counter these problems, we have developed a protocol that gives consistent readings that are fully representative of cell number while retaining some of the original advantages of soluble formazan assays.

Animals↗

PubMed1984

Improvement in soluble dehydrogenase histochemistry by nitroblue tetrazolium preuptake in sections: a qualitative and quantitative study.

An improvement in the histochemical demonstration of soluble dehydrogenase enzymes has been obtained by preincubating frozen sections in a nitroblue tetrazolium (NBT)/acetone solution, followed by routine incubation in polyvinyl alcohol (PVA) enriched media. Tissue binding properties of NBT were shown clearly to be decreased in histochemical media containing the colloid PVA for soluble enzymes, thus causing loss of the final reaction product (formazan) from the sections. The preincubation step in NBT/acetone allows tetrazolium salt to bind firmly to tissue lipoprotein (substantivity) and diminishes the loss of reduced formazan from heavily reacting tissue sections. The time course of NBT substantivity was examined and it was found that NBT binds rapidly to tissues (liver, kidney, heart) during preincubation, so that a preincubation of 30-60 seconds at room temperature is sufficient to improve the final morphological results greatly. Microspectrophotometric measurements of matched controls and NBT/acetone preincubated sections show that the preincubation step may slightly decrease lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) activities. This decrease was probably due to increased binding efficiency of formazan to cell lipoproteins but was judged, however, to be irrelevant compared to the morphological advantages produced by the NBT/acetone preincubation procedure.

Animals↗

PubMed1997

The tetrazolium dyes MTS and XTT provide new quantitative assays for superoxide and superoxide dismutase.

The tetrazolium dyes MTS and XTT were reduced to their soluble formazans by superoxide radical anions (O2-) produced by the oxidation of xanthine by xanthine oxidase under standard conditions. These reactions were compared to the well-known reductions of NBT and cytochrome c by the xanthine/xanthine oxidase system. Reduction of the dyes was completely inhibited by superoxide dismutase (SOD). Rate constants for the reaction of MTS and XTT with O2- were estimated at 1.3 +/- .1 x 10(5) M-1S-1 and 8.6 x 10(4) M-1S-1 respectively. The stable MTS and XTT formazans have high extinction coefficients in the visible range which enable sensitive detection and quantification of superoxide radicals, avoiding some of the problems inherent in assays based on production of the insoluble NBT formazan. MTS and XTT have considerable potential both for the quantitative assay of radical production in living tissues and for the assay of superoxide dismutase activity in tissue extracts. Implications for the interpretation of cell culture growth assays which employ these dyes are discussed.

Cytochrome c Group↗