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PubMed1995

Aqueous soluble tetrazolium/formazan MTS as an indicator of NADH- and NADPH-dependent dehydrogenase activity.

Recently a new tetrazolium was described for the use of monitoring cell viability in culture. This tetrazolium, commonly referred to as MTS [3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt], has the unusual property that it can be reduced to a water-soluble formazan. beta-Nicotinamide adenine dinucleotide/reduced (NADH) and beta-nicotinamide adenine dinucleotide phosphate/reduced (NADPH) are examples of physiologically important reducing agents. In cell-free studies, MTS was reduce to the soluble formazan in the presence of NADH and NADPH, and reaction were compared to those with dithiothreitol (DTT) or 2-mercaptoethanol (2-ME). The efficiency of these reactions was enhanced 1000-fold by the presence of phenazine methosulfate. Selectivity in the electron transfer from NADPH was slightly greater than NADH, and NADPH or NADH was much greater than the thiols DTT or 2-ME. Generation of either NADH or NADPH in solution by malate dehydrogenase or isocitrate dehydrogenase, respectively, was monitored by the MTS reduction reaction. The rate of formazan formation was comparable to the formation of NADH or NADPH. This system represents a useful tool for evaluating reaction kinetics in solutions of NAD- or NADP-dependent dehydrogenase enzymes, and these reactions can be performed in typical biological buffers containing reducing agents without significant interference to the MTS/formazan system.

Dithiothreitol↗
PubMed2001

Resveratrol inhibits the proliferation of normal human keratinocytes in vitro.

Resveratrol, a phytoalexin found in grapes and other plants, is a potent antioxidant, anti-inflammatory, and antiproliferative agent that is thought to have chemopreventive properties with respect to carcinogenesis. However, the antiproliferative effects of resveratrol have been described primarily for cultured tumor cells and its effects on the proliferation of normal cells are not clear. We evaluated the viability and proliferation of cultured normal human keratinocytes (KCs) exposed to resveratrol (0.25-100 microM) for different lengths of time (5-72 h) by means of (3)H-thymidine incorporation, direct cell counts, and a tetrazolium-based formazan reaction. The first two methods indicated that resveratrol, even at low concentrations, induced a time-and concentration-dependent inhibition of KC proliferation. However, formazan production was actually increased at moderate resveratrol concentrations (10 microM) and diminished only at higher concentrations. Even brief exposure (5 h) of KCs to resveratrol resulted in a concentration-dependent elevation in formazan production. This was blocked by ionomycin but was not dependent on Ca(2+). We conclude that resveratrol, even at submicromolar concentrations, inhibits the proliferation of normal human KCs in vitro and, at higher concentrations (40-100 microM), is cytotoxic to these cells. J. Cell. Biochem. Suppl. 36: 55-62, 2001.

Antineoplastic Agents↗
PubMed1994

Quantitative comparison between the gel-film and polyvinyl alcohol methods for dehydrogenase histochemistry reveals different intercellular distribution patterns of glucose-6-phosphate and lactate dehydrogenases in mouse liver.

The precise histochemical localization and quantification of the activity of soluble dehydrogenases in unfixed cryostat sections requires the use of tissue protectants. In this study, two protectants, polyvinyl alcohol (PVA) and agarose gel, were compared for assaying the activity of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) in normal female mouse liver. Quantification of enzyme activity was determined cytophotometrically in periportal (PP), pericentral (PC) and midzonal (MZ) areas. No coloured reaction product was present in PVA media after the incubation period. In contrast, the agarose gels appeared to be highly coloured after incubation. As a consequence, sections incubated with gel media were less intensely stained than those incubated in PVA-containing media. The specific G6PDH reaction (test minus control) yielded approximately 75% less formazan in sections incubated by the agarose gel method than with the PVA method. Further, the amount of formazan deposits attributable to G6PDH activity was highest in the midzonal and pericentral zones of the liver lobule with PVA media, and Kupffer cells could be discriminated easily because of their high G6PDH activity. Significant zonal differences or Kupffer cells could not be observed when agarose gel films were used for the detection of G6PDH activity. The LDH localization patterns appeared to be more uniform after incubation with both methods: no significant differences in specific test minus control reactions were seen between PP, PC and MZ. However, less formazan production (33%) was detected in sections incubated with agarose gels when compared with those incubated with PVA media.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Histochemical demonstration of sodium-dependent glutamate uptake in brain tissues by glutamate dehydrogenase reaction.

Using a special tetrazolium salt technique, a striking correlation was observed between Na+ concentration of the incubation medium and the formation of formazan catalyzed by glutamate dehydrogenase (GDH) in glutamatergic neuropil areas of the hippocampal formation, cerebellum, and other brain regions. Na+ concentrations of 130-150 mmol/l caused maximal formazan production. The GDH catalyzed sodium-dependent increase in formazan production is suggested to be a consequence of the sodium dependence of glutamate uptake in glutamatergic brain structures supplying the enzyme with substrate.

Animals↗
PubMed1978

The measurement of histochemically-stained NADP-dependent isocitrate dehydrogenase in the sebaceous glands of hairless hamster.

The effect of substrate, co-factor and Nitroblue Tetrazolium concentration on the production of formazan by the action of NADP-dependent isocitrate dehydrogenase was studied in the sebaceous glands of the hairless hamster. The measurement (in average optical density units per unit area) of formazan in histochemically stained skin sections was carried out by television scanning microdensitometry (Quantimet 720D). Having established optimal conditions for this enzyme, a second study was carried out to determine the effect of different power objectives and wide-band wavelength light instead of white light on the average optical density per unit area, recorded by the instrument, of the formazan produced in a defined number of sebaceous glands in a single skin section. It was found that there was no difference in the average optical density per unit area recorded by the instrument at different power objectives and a peak value of average optical density per unit area could be obtained using a K4 or a K5 Balzer filter (550-650 nm).

Animals↗
PubMed1992

Islet cell metabolism is reflected by the MTT (tetrazolium) colorimetric assay.

Insulin secretion depends critically on glucose metabolism. We investigated whether a rapid viability test could be established for assessing glucose metabolism in insulin secreting cells. The MTT (C,N-diphenyl-N'-4,5-dimethyl thiazol-2-yl tetrazolium bromide) colorimetric assay (reduction of tetrazolium salt to formazan) was applied to rat islets and rat insulinoma cell lines. It was found that the rate of formazan production correlated with glucose oxidation and glucose utilization at glucose concentrations which also stimulated insulin secretion. In differentiated insulinoma INS-1 cells, salt reduction paralleled the insulin release at glucose concentrations of up to 8.3 mmol/l. The glucose-induced formazan production in INS-1 cells and islets was abolished by exposure to the Beta-cell cytotoxic agents, streptozotocin or alloxan. The MTT assay thus provides a convenient tool for the rapid assessment of Beta-cell metabolism and viability.

Alloxan↗
PubMed2005

An evaluation of three new-generation tetrazolium salts for the measurement of respiratory activity in activated sludge microorganisms.

XTT (3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt), and WST-1 (4-(3-4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzenedisulfonate) are tetrazolium salts that have become commercially available only in relatively recent years; they differ from earlier such compounds in that their reduction gives rise to a formazan product that is water soluble. We have established the sites in the prokaryotic respiratory chain at which each of the dyes is reduced to its corresponding formazan and have evaluated the suitability of each for the colorimetric estimation of electron transport system activity in populations of activated sludge microorganisms. Reduction of all three tetrazolium salts was shown to be proportional to cell biomass and oxygen uptake and to be susceptible to low levels of the reference toxicant 3,5-dichlorophenol. XTT, which was not inhibitory at concentrations of up to 2 mM and was reduced by 91% of isolates from a sample of culturable activated sludge bacteria, was chosen for further assay development. XTT-formazan production was found to be stimulated by the availability of an exogenous carbon and energy source, and by the presence of the electron-coupling agent phenazine methosulfate. Less than 3% of XTT reduction by an activated sludge sample was abiotic. An assay based on this compound could be a valuable and simple tool for the routine monitoring of the performance of wastewater treatment systems.

Bacterial Physiological Phenomena↗
PubMed1988

The amplified detection of free and bound methoxatin (PQQ) with nitroblue tetrazolium redox reactions: insights into the PQQ-locus.

Porcine kidney diamine oxidase, a PQQ-enzyme, can be directly measured by formazan production with putrescine and nitroblue tetrazolium. This cyclic reaction in air is unaffected by superoxide dimutase, suggesting a two electron transfer between substrate-reduced PQQ-locus and nitroblue tetrazolium, without intermediate formation of superoxide. With albumin-bound PQQ and detergent-exposed PQQ-loci, glycine can be oxidized by PQQ and electrons repetitively transferred through PQQ-sites to nitroblue tetrazolium, the rate of formazan production detecting picomoles of exposed PQQ-locus. Exposed PQQ-loci are also reducible with NaCNBH3. Nitroblue tetrazolium, reoxidizes the reduced PQQ-locus with formazan production. These experiments suggest that the PQQ-locus of quinoproteins contains a [ketone-ketoimine in equilibrium with ketoamine] redox center.

Amine Oxidase (Copper-Containing)↗
PubMed1984

Pathways of hydrogen utilization from NADPH generated by glucose-6-phosphate dehydrogenase in circumventricular organs and the hypothalamo-neurohypophysial system: a cytochemical study.

Cytochemistry was used to examine the distribution of two pathways of utilization of hydrogen (Type I and Type II H) generated by glucose-6-phosphate dehydrogenase (G6PD) in circumventricular organs (CVOs) and the hypothalamo-neurohypophysial system in cryostat sections of rat brain. Type I H is defined as that portion of the total reducing equivalents (Total H) that is passed, in the intact cells, along the cytochrome chain (NADPH-diaphorase system). In the liver, energy from Type I H is used for cytochrome P-450-dependent oxidation of steroids, as well as xenobiotics. We proposed that mixed function oxidation, and therefore Type I H, would be preferentially localized in brain regions lacking a blood-brain barrier, such as CVOs and magnocellular cells with terminals in such brain regions. Type I H was identified in tissue sections using neotetrazolium. This reagent, when reduced, precipitates as formazan granules that can be quantified. The large difference in redox potential between NADPH and neotetrazolium ensures that only hydrogen (Type I H) passed in the intact cell along the cytochrome chain, can reduce the tetrazole. Total NADPH generation (Total H) from glucose-6-phosphate, was identified using medium containing phenazine methosulphate, a hydrogen acceptor that transfers all reducing equivalents from NADPH to the tetrazole. Type II H, the difference between Total and Type I H, is presumed to be used for NADPH-dependent biosynthetic functions such as lipid synthesis, or reduction of glutathione. In CVOs formazan granules indicative of Type I H were selectively concentrated and localized within cells throughout the SFO, organum vasculosum of the lamina terminalis, pineal gland and in the apical cytoplasm of columnar ependymocytes in the subcommissural organ. Formazan granules attributable to Type I H were also prominent throughout the hypothalamo-neurohypophysial system. Reaction product was present in the cytoplasm of some magnocellular neurons in both the supraoptic and paraventricular nuclei, in the median eminence, including the zona interna, and in and between cells in the neurohypophysis. The distribution of NADPH-diaphorase in sections incubated with NADPH instead of glucose-6-phosphate was similar to that of Type I H. These findings are consistent with the hypothesis that mixed function oxidation involving NADPH and the cytochrome chain occur in these brain regions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Visualization of reactive oxygen species formation by phagocytizing macrophages.

Activated peritoneal macrophages exhibiting phagocytosing capacity produced an electron-dense precipitate of formazan in contact sites of macrophage plasmalemma and phagocytosed yeast cells. No production of formazan occurred, when non-opsonized latex particles were ingested by macrophages. Formazan precipitation could be prevented by anaerobiosis but not by addition of cyanide.

Anaerobiosis↗
PubMed1989

Colorimetric quantitation of filarial viability.

A simple three-step colorimetric assay based on the tetrazolium salt MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) has been developed for quantifying filarial viability. Living (but not dead) filariae take up MTT and rapidly reduce it to formazan, so staining themselves dark blue. This colour change which is easily seen provides a rapid qualitative test for filarial viability. Quantitative data can be obtained by solubilizing formazan out of the worm with DMSO and measuring the absorbance of the resulting solution at 510 nm. To date the technique has been demonstrated in several species of filariae including Onchocerca volvulus. MTT reduction is thought to be selective for NADH-dependent dehydrogenase activity in viable worms. The reaction occurs readily in all developmental stages of Dipetalonema viteae including fragments of filarial tissue. Enzyme activity in viable intact D. viteae appears to be primarily associated with the hypodermis/muscle cells, with minimal formazan formation in the gut and reproductive tracts. The application of this MTT assay as a parameter for quantifying in vitro drugs effects is described. Assay procedures have been developed and optimized with D. viteae and Brugia pahangi for the assessment of effects of macrofilariae and microfilarial release, and the activity of a range of antifilarial standards reported. Several potential applications of the technique to studies on filarial biology are discussed.

Animals↗
PubMed1990

MTT colorimetric assay for testing macrophage cytotoxic activity in vitro.

The MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) cleavage assay, originally described by Mosmann (1983, J. Immunol. Methods 65, 55) for measuring cell survival/proliferation, has been used successfully to quantitate macrophage-mediated cytotoxicity. Peritoneal macrophages, from control or pyran copolymer MVE2-treated mice and from TU5 and L929 murine cell lines, were used as effectors and targets respectively in a 48 h cytotoxicity test, at three effector: target ratios (10:1, 5:1, 2:1). The amount of MTT reduced by cells to its blue formazan derivative during an additional 4 h of culture was quantified spectrophotometrically at 570 nm using an ELISA reader. A linear relationship between the formazan generated and the number of viable TU5 and L929 cells was demonstrated, together with time-dependent growth characteristics for these cells. The formazan produced by macrophages was independent of their functional state and did not interfere with the target cell signal. MVE2-activated macrophages strongly inhibited the survival/growth of target cells in a dose-related way, whereas the cytotoxic activity of control macrophages was very low. Finally, the MTT method compared favorably with the 3H-TdR uptake method in evaluating macrophage cytotoxicity, and both of them were more sensitive than the 3H-TdR release assay. The MTT cleavage method is a useful alternative to radioisotopic methods for quantitating macrophage cytotoxicity for actively growing in vitro targets. Its main advantages are: (a) sensitivity and reproducibility; (b) elimination of the need for radioactive compounds; (c) ease with which it can be performed and quantified; (d) rapidity.

Animals↗
PubMed1991

An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT.

A new tetrazolium salt XTT, sodium 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6- nitro)benzene-sulfonic acid hydrate, was evaluated for use in a colorimetric assay for cell viability and proliferation by normal activated T cells and several cytokine dependent cell lines. Cleavage of XTT by dehydrogenase enzymes of metabolically active cells yields a highly colored formazan product which is water soluble. This feature obviates the need for formazan crystal solubilization prior to absorbance measurements, as required when using other tetrazolium salts such as MTT. Bioreduction of XTT by all the murine cells examined was not particularly efficient, but could be potentiated by addition of electron coupling agents such as phenazine methosulfate (PMS) or menadione (MEN). Optimal concentrations of PMS or MEN were determined for the metabolism of XTT by the T cell lines HT-2 and 11.6, NFS-60 a myeloid leukemia, MC/9 a mast cell line and mitogen activated splenic T cells. When used in combination with PMS, each of these cells generated higher formazan absorbance values with XTT than were observed with MTT. Thus the use of XTT in colorimetric proliferation assays offer significant advantages over MTT, resulting from reduced assay time and sample handling, while offering equivalent sensitivity.

Animals↗
PubMed1995

Microculture tetrazolium assays: a comparison between two new tetrazolium salts, XTT and MTS.

Microculture tetrazolium assays are being widely exploited to investigate the mechanisms of both cell activation and cell damage. They are colorimetric assays which are based upon the bioreduction of a tetrazolium salt to an intensely coloured formazan. We contrast the responses obtainable with two new tetrazolium salts, MTS and XTT, when used on the rat lymphoma cell line (Nb2 cells), which has been activated by human growth hormone. These tetrazolium salts, unlike the more commonly used MTT, form soluble formazans upon bioreduction by the activated cells. This has the advantage that it eliminates the error-prone solubilisation step which is required for the microculture tetrazolium assays which employ MTT. Bioreduction of XTT and MTS usually requires addition of an intermediate electron acceptor, phenazine methosulphate (PMS). We found that the XTT/PMS, but not the MTS/PMS, reagent mixture was unstable. Nucleation and crystal formation in the XTT/PMS reagent mixture, prepared in DPBS, could occur within 1-3 min. This resulted in a decline in XTT-formazan production and manifested itself in the microculture tetrazolium assay as both poor within-assay precision and serious assay drift. Several features of the system suggested that the formation of charge-transfer complexes between XTT and PMS accounted for this instability. No such instability was encountered when MTS and PMS were mixed. We demonstrate that MTS/PMS provides microculture tetrazolium assays for hGH which are free from these serious artefacts and which are uniquely precise. In conclusion we therefore advocate the use of MTS in preference to XTT for the new generation of microculture tetrazolium assays.

Animals↗
PubMed2005

Modification of MTT assay conditions to examine the cytotoxic effects of amitraz on the human lymphoblastoid cell line, WIL2NS.

Reported parameters of the MTT assay vary widely, and reflect a need to optimise the assay for different cell types. The MTT assay conditions for the human B-lymphocyte-derived cell line WIL2NS were optimised for MTT incubation and formazan development. The optimised MTT assay was validated by examining the effects of the acaride amitraz on WIL2NS. In pH-buffered media in the absence of cells, MTT formed formazan spontaneously, and absorbance was proportional to both the initial concentration of MTT and the time of incubation at 37 degrees C. One milligram per millilitre MTT was toxic to WIL2NS cells, but the accuracy of the standard curve was reduced when only 0.2 mg/ml MTT was used. Twenty percent SDS in 0.2 M HCl was preferable to DMSO as a solvent for formazan. Exposure to 0.035% amitraz resulted in a significant reduction in WIL2NS cell numbers after only 2 h of exposure. It was concluded that 0.035% of amitraz has the potential to adversely affect lymphocytes in the systemic blood system in humans, and that an optimised MTT assay was obtained by incubating WIL2NS cells with 0.45 mg/ml MTT for 17 h, followed by addition of acidified SDS for 1 h.

Cell Line↗
PubMed1995

Demonstration of nitric oxide synthase (NOS) in marmosets by NADPH diaphorase (NADPH-d) histochemistry and NOS immunoreactivity.

Since species interdiversity often prevents the extrapolation of laboratory rodent data to man and similar problems may exist for nitric oxide synthase (NOS), NADPH-d activity and immunohistochemistry of NOS were investigated in the New World monkey Callithrix jacchus (marmoset), which has been shown to be close to the human situation in many respects. Using the NADPHd reaction with beta-NADPH and nitroblue tetrazolium (NBT) on acetone-chloroform pretreated cryosections, NBT formazan was found in many neural and non-neural (e.g. diverse epithelia, striated muscle fibers, vascular endothelium) cells in numerous tissues and organs. Prefixation with formaldehyde lowered the number of NADPH-d active sites and the amount of formazan with the exception of neuronal NADPH-d as did incubation of fresh or acetone-chloroform-pretreated sections for NADPH-d in the presence of 0.5% formaldehyde. When 1% formaldehyde or 0.5 mM permanganate were used significant amounts of formazan appeared only in central and peripheral neurons, vasal endothelial cells, small intestinal enterocytes, plasma membrane region of striated muscle fibers as well as arteriolar cells in the kidney; except for enterocytes, these observations were confirmed by NOS-immunohistochemistry which revealed in addition reactive cells in the thymus and intestinal lamina propria.

Acetone↗
PubMed1999

Application of a tetrazolium dye as an indicator of viability in anaerobic bacteria.

The use of the redox dye 5-cyano-2,3,-ditolyl tetrazolium chloride (CTC) for evaluating the metabolic activity of aerobic bacteria has gained wide application in recent years. In this study, we examined the utility of CTC in capturing the metabolic activity of anaerobic bacteria. In addition, the factors contributing to abiotic reduction of CTC were also examined. CTC was used in conjunction with the fluorochrome 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF), that targets bacterial cell wall proteins, to quantitate the active fraction of total bacterial numbers. Facultative anaerobic bacteria, including Escherichia coli grown fermentatively, and Pseudomonas chlorophis, P. fluorescens, P. stutzeri, and P. pseudoalcalegenes subsp. pseudoalcalegenes grown under nitrate-reducing conditions, actively reduced CTC during all phases of growth. Greater than 95% of these cells accumulated intracellular CTC-formazan crystals during the exponential phase. Obligate anaerobic bacteria, including Syntrophus aciditrophicus grown fermentatively, Geobacter sulfurreducens grown with fumarate as the electron acceptor, Desulfovibrio desulfuricans subsp. desulfuricans and D. halophilus grown under sulfate-reducing conditions, Methanobacterium formicicum grown on formate, H2 and CO2, and Methanobacterium thermoautotrophicum grown autotrophically on H2 and CO2 all reduced CTC to intracellular CTC-formazan crystals. The optimal CTC concentration for all organisms examined was 5 mM. Anaerobic CTC incubations were not required for quantification of anaerobically grown cells. CTC-formazan production by all cultures examined was proportional to biomass production, and CTC reduction was observed even in the absence of added nutrients. CTC was reduced by culture fluids containing ferric citrate as electron acceptor following growth of either G. metallireducens or G. sulfurreducens. Abiotic reduction of CTC was observed in the presence of ascorbic acid, cysteine hydrochloride, dithiothreitol, NADH, NADPH, Fe(II)Cl2, sodium thioglycolic acid and sodium sulfide. These results suggest that while CTC can be used to capture the metabolic activity of anaerobic bacteria, care must be taken to avoid abiotic reduction of CTC.

Bacteria, Anaerobic↗
PubMed1999

Enhancement of MTT, a tetrazolium salt, exocytosis by amyloid beta-protein and chloroquine in cultured rat astrocytes.

The effect of amyloid beta-protein (Abeta) on the cellular reducing activity has been a controversial issue. We determined the cellular reducing activity in cultured astrocytes using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl )-2H-tetrazolium (WST-8) reduction assays following Abeta treatment. MTT reduction was inhibited whereas WST-8 reduction was unaffected by the Abeta treatment. Furthermore, the early extracellular appearance of MTT formazan, a reduced product of MTT, was observed in association with the rapid disappearance of intracellular formazan granules. Notably, similar results were obtained in cultures treated with chloroquine, a perturbant of membrane trafficking. Our results suggest that MTT formazan exocytosis is enhanced in a similar manner by Abeta and chloroquine and that this biological activity of Abeta may underlie the pathogenesis of Alzheimer's disease.

Amyloid beta-Peptides↗