Water-soluble Formazan Research Articles

Is reduction of the sulfonated tetrazolium 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2-tetrazolium 5-carboxanilide a reliable measure of intracellular superoxide production?

The sulfonated tetrazolium 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2-tetrazolium 5-carboxanilide (XTT) is advantageous in that it yields a water-soluble formazan, unlike most other available tetrazoliums. XTT is reducible by superoxide, as are other tetrazoliums, but is not directly reduced by xanthine oxidase plus xanthine or by glucose oxidase plus glucose. This led to the suggestion that XTT reduction might serve as a reliable index of intracellular O 2 − production. We now show that soluble extracts of Escherichia coli contain two NADPH:XTT reductases that act aerobically or anaerobically. That being the case, XTT reduction is not a reliable measure of intracellular O 2 −.

Spectrophotometric assay of superoxide anion formed in Maillard reaction based on highly water-soluble tetrazolium salt.

A novel highly water-soluble tetrazolium salt, WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt), can be reduced to water-soluble formazan with a superoxide anion. Here, the WST-1 assay was applied to detect the superoxide anion generated during the Maillard reaction. A sample solution containing carbonyl compounds such as glyceraldehyde and glycolaldehyde (5 mM) and Na-acetyl-L-lysine (10 mM) was incubated for 2 days at 37 degrees C. The detection of a superoxide anion generated in the sample was performed by the WST-1 assay, and the result was compared with the cytochrome c assay. The reduction of WST-1 was almost perfectly (86-96%) inhibited by the addition of the superoxide dismutase (SOD). On the contrary, the reduction of cytochrome c was slightly (20-25%) inhibited by the addition of SOD. A similar result was observed for the addition of 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron). These results mean that the specificity of WST-1 to the superoxide anion was superior to that of cytochrome c. It was also possible to continuously monitor superoxide anion generation during the Maillard reaction by the coexistence of WST-1 in the reaction solution.

XTT formazan widely used to detect cell viability inhibits HIV type 1 infection in vitro by targeting gp41.

XTT can be metabolically reduced by mitochondrial dehydrogenase in viable cells to a water-soluble formazan product. Thus XTT has been widely used to evaluate cell viability and to screen anti-HIV agents and the cytotoxicity of these agents. The present studies demonstrated that XTT formazan derived from XTT in cell culture significantly inhibits the fusion of HIV-1-infected cells with uninfected cells. Synthetic XTT formazan effectively inhibited the replication of laboratory-adapted and primary HIV-1 isolates and cell-to-cell fusion with low cytotoxicity. It blocks the six-helix bundle formation between peptides derived from the N- and C-terminal heptad repeat regions of the gp41 ectodomain (designated N- and C-peptides, respectively). Analysis by a computer-aided docking program indicates that XTT formazan may bind to the highly conserved hydrophobic pocket on the surface of the central trimeric coiled coil of gp41. These results suggest that XTT formazan inhibits HIV-1 entry by targeting the alpha-helical coiled-coil domain of gp41. This small molecular nonpeptide antiviral compound can be used as a lead for designing more effective HIV-1 entry inhibitors targeting the fusion stage of HIV-1 infection. But because XTT formazan itself has anti-HIV-1 activity, caution should be exercised when XTT is used to evaluate HIV-1 infectivity.

Development of a synchronous enzyme-reaction system for a highly sensitive enzyme immunoassay.

A synchronous enzyme-reaction system using water-soluble formazan and a non-enzymatic electron mediator was developed and applied to an enzyme immunoassay (EIA). The reaction system consists of four steps: (I) dephosphorylation of NADP(+) to produce NAD(+) by alkaline phosphatase (ALP), (II) reduction of NAD(+) to produce NADH with oxidation of ethanol to yield acetaldehyde by alcohol dehydrogenase (ADH), (III) reduction of water-soluble tetrazolium salt (WST-1) to produce formazan by NADH via 1-methoxy-5-methyl-phenazinium methyl sulfate (PMS), and (IV) re-reduction of NAD(+) to produce NADH by ADH. During each cycle, one molecule of tetrazolium is converted to one molecule of formazan. The concentration of formazan during the reaction was given by second-order polynomials of the reaction time. Kinetic studies strongly suggested that the synchronous enzyme-reaction system had the potential to detect an analyte at the attomole level in EIA. On the basis of the kinetic studies, optimal conditions for EIA incorporating the synchronous system were examined. NADP(+) was purified thoroughly to remove minor traces of NAD(+) in the preparation, and an ADH preparation contaminated with the lowest level of ALP activity was used. When the synchronous system was applied to a sandwich-type EIA for human C-reactive protein, the protein was detected with a sensitivity of 50 attomole per well of a micro-titer plate (0.1 ml) in a 1-h reaction. In addition, EIA with water-soluble formazan showed a more quantitative and sensitive result than that with insoluble formazan. These findings indicated that the (WST-1)-PMS system introduced in this study has a great potential for highly sensitive enzyme immunoassay.

Superoxide produced by activated neutrophils efficiently reduces the tetrazolium salt, WST-1 to produce a soluble formazan: a simple colorimetric assay for measuring respiratory burst activation and for screening anti-inflammatory agents

Activation of the respiratory burst of granulocytes and macrophages by invading microorganisms is a key first line cellular defence against infection. Failure to generate this response leads to persistent life-threatening infection unless appropriate antibiotic treatment is given. The respiratory burst of neutrophils is usually measured spectrophotometrically by following ferricytochrome c reduction, and histologically by using the tetrazolium salt, nitroblue tetrazolium, which is reduced intracellularly to an insoluble formazan. In both assays, reduction is mediated by superoxide generated via NADPH oxidase. Because ferricytochrome c has a high molecular mass and high background absorbance at 550 nm, the assay lacks sensitivity and is not ideally suited to microplate measurement. We have circumvented these limitations by using the cell-impermeable, sulfonated tetrazolium salt, WST-1, which exhibits very low background absorbance and is efficiently reduced by superoxide to a stable water-soluble formazan with high molar absorptivity. This has permitted adaptation of the WST-1 assay to microplate format while retaining sensitivity. Reduction of WST-1 by activated human peripheral blood neutrophils correlated closely with ferricytochrome c reduction across a range of PMA concentrations and with time of activation by PMA and fMLP. Reduction of WST-1 was inhibited by 98% by superoxide dismutase (20 μg/ml) and by 88% by the NADPH oxidase inhibitor, diphenyleneiodinium (10 μM) but was resistant to catalase, azide and the NADH oxidase inhibitor, resiniferatoxin. WST-1 and ferricytochrome c reduction were also compared using xanthine/xanthine oxidase to generate superoxide. Under optimised assay conditions, both WST-1 and ferricytochrome c reduction were directly proportional to added xanthine. WST-1 generated approximately 2-fold greater increase in absorbance than ferricytochrome c at their respective wavelengths, and this translated into increased assay sensitivity. Addition of the intermediate electron acceptor, 1-methoxy phenazine methosulfate, increased the background of the neutrophil assay but did not affect the overall magnitude of the response. We have used the WST-1 assay to assess human neutrophil dysfunction and to compare anti-inflammatory activity.

Routine Assessment of Viability in Split-Thickness Skin

Effective quality control of allograft skin that is cryopreserved for transplantation requires a simple, reproducible technique for the assessment of cell viability. Tetrazolium reduction assays and an oxygen consumption technique have been the two methods of choice to determine the metabolic function of allograft skin after it has been thawed. In this study, we investigated the use of a novel tetrazolium salt, WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzen e disulfonate), that is cleaved to a water-soluble formazan product. Porcine split-thickness skin in minimal essential medium without cryoprotectant was subjected to a graded freezing protocol to generate progressive amounts of cryoinjury. Recovery as determined with WST-1 was compared with measurements made with the use of the oxygen consumption technique. The similarity of the resulting recovery curves indicates that WST-1 is a simple, effective, and convenient technique for the assessment of metabolic function in porcine split-thickness skin. The WST-1 assay is applicable for the routine assessment of tissue viability in cryopreserved allograft skin.

In vitro toxicity of surfactants in U937 cells: Cell membrane integrity and mitochondrial function

Surface-active agents are components of many drugs and cosmetics. In order to examine their cytotoxic and membrane-toxic potential, various surfactants were examined in U937 cells using the tetrazolium reduction assay EZ4U, a modified XTT test, and the arachidonic acid release test (AART). EZ4U measures the ability of living cells to reduce a colorless tetrazolium salt to an orange water-soluble formazan derivative by mitochondrial dehydrogenases. [3H]arachidonic acid ([3H]AA) is rapidly incorporated into cell membrane phospholipids. Due to membrane disintegration or enzymatic catalysis, it is released into the cell culture medium and can be measured by scintillation technique. The results after 24-hour-exposure are as follow (CC50 microg/ml; RC50 microg/ml): benzalkonium chloride (0.6), Cremophor A25 (1.4; 5.9), sodium cetearyl sulfate (1.6; 19.9), Brij78 (1.6; 7.7), TEGO betaine E (3.0; 19.3), TEGO betaine CKD (4.1; 20.2), TEGO betaine L7 (7.5; 20.0), sodium dodecyl sulfate (7.5; 39.0), Triton X-100 (8.9; 110), polysorbate 80 (48.1; 491), soybean lecithin (7920; 19940).

Toxicity and Cell Density Monitoring in Monolayer and Three-dimensional Cultures with the XTT Assay

The application of viability criteria (MTT and XTT tests) to monolayer cultures and immobilised cells in three-dimensional systems was investigated in order to assess cell viability and cell proliferation. The suitability and accuracy of these tests were compared with the conventional criteria (cellular protein and DNA content) used in monolayer cultures for the same purpose. The colorimetric assay based on the metabolic reduction of the tetrazolium salt XTT to a water-soluble formazan proved to be very useful, rapid and sensitive. This automated spectrophotometric enzymatic method, due to its lack of toxicity, also permits repeated nondestructive assays on a single cellular culture for the long-term monitoring of cytotoxicity, cell survival and cell proliferation, and can be performed in 96-well plates with minimal handling. This method could offer a solution for cellular density evaluation in complex cell cultures that do not permit visual examination; it is also the best choice for protein-based, three-dimensional systems such as collagen gels.

Enzymatic method for assaying uric acid in serum with a new tetrazolium salt produces water-soluble formazan dye.

To apply an enzymatic method for assaying uric acid in serum based on the uricase (EC 1.7.3.3)-catalase (EC 1.11.1.6)-formaldehyde dehydrogenase (FADH, EC 1.2.1.46) coupled with the new tetrazolium salt producing a water-soluble formazan dye as an indicator system. Unlike the traditional tetrazolium salts, e.g., iodonitrotetrazolium (INT) and nitrotetrazolium blue (NTB), the corresponding formazan dye produced did not absorb to the test tube and the reaction cells of the analyzer. Moreover, this formazan dye had a higher water solubility and more sensitivity. A two electron reduction of tetrazolium salt with NADH, produced by the uricase-catalase-FADH reaction, was mediated by an electron carrier, 1-methoxy PMS. The generation of formazan, monitored at 440 nm, was proportional to the concentration of uric acid in serum. The sensitivity of this method was about 1.5 times that of the peroxidase-TOOS [N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine] method. The assay was evaluated with TBA-80FR.NEO biochemical analyzer. The average within-run and between-day imprecision (CV) were 0.75-2.44% and 3.20-3.33%, respectively. Results of the proposed method (y) correlated well with those determined by the conventional chromogen method (x): y = 0.970 x - 0.018 mmol/L (Sy l x = 0.019 mmol/L, r = 0.993, n = 67). We also present data showing that the method is rapid, relatively free of interference, and amenable to automation.

Evaluation of MTS, XTT, MTT and3HTdR incorporation for assessing hepatocyte density, viability and proliferation

The new 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) colorimetric assays for assessing hepatocyte density, viability and proliferation were evaluated and compared with 3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and tritiated thymidine (3HTdR) incorporation. OD values of MTS or XTT, which are metabolically reduced in viable cells to a watersoluble formazan product and can be read directly, had a good correlation coefficient with hepatocyte densities in a range of 2.5–40×104 cells/ml (MTSr=0.952; XTTr=0.902) and with hepatocyte viability (MTSr=0.974; XTTr=0.975). At 0, 20, 40 and 60 hr cultures, the correlation coefficients with3HTdR for assessing hepatocyte viability and proliferation (MTSr=0.942–0.981; XTTr=0.953–0.992) were excellent. In contrast with MTS and XTT, MTT OD values had a poor correlation coefficient with hepatocyte densities (r=0.672), viability (r=0.622) and3HTdR incorporation (r=0.701–0.818 at 0, 20 and 40 hour cultures). This study shows that the MTS or XTT colorimetric assays for assessing hepatocyte density, viability and proliferation are more accurate, reliable and simpler than the widely used MTT assay. They avoid use of radioactive material as required for3HTdR incorporation. Of the two, the MTS colorimetric assay is more sensitive than the XTT.

A simple quantitative assay for the killing of in vitro culture cells by Ehrlichia canis infection

A simple colorimetric assay, on the basis of bioreduction of the substrate XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt) by dehydrogenases of actively metabolizing mammalian cells, was assessed for measurement of in vitro killing of canine macrophage-like cells by Ehrlichia canis infection. Formation of a highly colored, water soluble formazan which resulted from bioreduction by the test cells was significantly enhanced by phenazine methosulfate, an electron coupling agent. Normal cells markedly reduced XTT in the presence of phenazine methosulfate, yielding a post-/pre-bioreduction absorbance ratio of >4.0 under the assay conditions tested. E. canis-infected cells, however, markedly lost the capability for bioreduction, producing a post-/pre-bioreduction absorbance ratio as low as 2.0. Colorimetric assay results correlated with E. canis infections as tested by an indirect fluorescent antibody assay ( R = 0.95). This colorimetric assay involved a single step and results could be obtained in 1 h. Thus, the microtiter colorimetric assay was able to quantitate killing of the cells by E. canis infection with the advantages of reliability and simplicity. It will also prove valuable in facilitating the development of other in vitro assays in ehrlichiosis research.

Resting-cell dehydrogenase assay measuring a novel water-soluble formazan detects catabolic differences among cells

A resting-cell assay using the novel tetrazolium compound, 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethyl-2-thiazolyl)-3-(4-sulfophenyl)tetrazolium, inner salt (MTS), demonstrates the presence of dehydrogenases in cells. When used with the electron-coupling reagent phenazine methosulfate (PMS), MTS can be reduced by cells into a water-soluble formazan. This assay detects different catabolic activities of cells grown with various carbon sources. In this study, the effect of repression of metabolic pathways was demonstrated in Escherichia coli JL3676, Bacillus subtilis ATCC 6633, and Pseudomonas stutzeri JM300 by assaying for formazan formation. Lactose-grown E. coli substrate as compared to glucose-grown or glucose-plus-lactose-grown cells. In addition, when compared to glucose-grown or glucose-plus-oleate-grown cells, oleate-grown E. coli cells showed at least a twenty-fold increase in activity when oleate was the added substrate. When succinate was the added substrate, succinate-grown B. subtilis cells produced a thirteen-fold increase in activity over glucose-grown or glucose-plus-succinate-grown cells. When glucose was the added substrate, glucose-grown P. stutzeri cells demonstrated at least a twelve-fold increase in activity compared with succinate-grown or succinate-plus-glucose-grown cells. Also, results comparable to the production of [ 14C]CO 2 from [ 14C]oleate by E. coli were obtained demonstrating that the assay is an alternative to the use of radiolabeled substrates.

A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet.

Cell viability and in vitro cytotoxicity assay methods were developed using a combination of dyes, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1), neutral red (NR) and crystal violet (CV), with HeLa cells as a bioindicatior. As WST-1 produces a highly water soluble and non-cytotoxic formazan dye, ti allows each assay to be carried out in one culture dish. The combined cell viability assay using WST-1, NR and CV gave an absorbance that correlated linearly with the number of cells over the range 1000 to 50,000 cells/well. The combined assay was applied to the evaluation of IC50 values for sodium dodecyl sulfate as a model toxicant, which yielded similar values to those obtained with each assay independently.

MTS interferon assay: a simplified cellular dehydrogenase assay for interferon activity using a water-soluble tetrazolium salt.

MTS, a tetrazolium dye, is reduced by hydrogenases in living cells to a water-soluble formazan. When it is added to the medium at the end of a cytopathic effects (CPE) inhibition interferon assay, the formazan formed diffuses into the medium; the resultant optical density directly and quantitatively measures how much cellular damage has been produced by the challenge virus in the presence of different amounts of interferon. The use of MTS has considerable advantages in that after it is added, no further steps, such as washing of the cells, extraction of dye, or other manipulations, are needed.

Novel disulfonated tetrazolium salt that can be reduced to a water-soluble formazan and its application to the assay of lactate dehydrogenase

A new tetrazolium salt, 4-[3-(4-iodophenyl)-2-(2,4-dinitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate, sodium salt, that produces a highly water-soluble formazan dye upon reduction by reduced nicotinamide adenine dinucleotide (NADH) was synthesized. The reduction of the compound by NADH at a neutral pH is fast owing to its small reduction potential. The applicability of the compound to the assay of lactate dehydrogenase is described in comparison with a prevalent tetrazolium salt.

Application of a Tetrazolium Salt with a Water-Soluble Formazan as an Indicator of Viability in Respiring Bacteria

The tetrazolium salt sodium 3'-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis (4-methoxy-6-nitro)benzene-sulfonic acid hydrate (XTT) was examined for use as a colorimetric indicator of viability in respiring bacteria. XTT was reduced to an orange, water-soluble formazan product by Methylosinus trichosporium OB3b, Pseudomonas putida, Escherichia coli, and Bacillus subtilis. Formazan production was proportional to live cell biomass, and XTT was reduced by all cultures in the absence of added electron-coupling agents. XTT reduction by M. trichosporium OB3b was linear over several hours and was stimulated by the presence of an exogenous substrate (methanol). Addition of cyanide to cultures incubated under oxic conditions gave an initial 10-fold increase in XTT reduction. Viability of bacteria incubated in the absence of exogenous carbon substrates was measured as XTT reduction and compared with viability estimates from plate counts. Results obtained with the two methods were generally comparable, but the XTT assay was superior when cell recovery on plates was low. Incubation of E. coli for 7 days in the absence of exogenous carbon substrates decreased viability by 90%, whereas the corresponding decreases for cultures of M. trichosporium OB3b, P. putida, and B. subtilis were less than 40%.

Rapid colorimetric bioassay for screening of Fusarium mycotoxins.

The cytotoxicity of Fusarium mycotoxins was evaluated using a trichothecene sensitive cell line (BHK-21, baby hamster kidney cells) in combination with the MTT-cleavage test as an end-point measurement. Cells tended to be more sensitive to the type A trichothecenes with midpoint cytotoxicity values ranging from 1.6 ng/ml for T-2 toxin to 60 ng/ml for scirpentriol. The cytotoxicity value for deoxynivalenol (type B) was 112 ng/ml. The inherent disadvantage of the MTT-assay (formation of insoluble formazan) was overcome by using the analog MTS and measuring the water-soluble formazan directly in the culture media. The MTS-midpoint cytotoxicity values for T-2 toxin and deoxynivalenol (2.1 and 141 ng/ml, respectively), although slightly higher, showed a good correspondence to the MTT-test. Both the MTT- and MTS-cleavage tests are useful for evaluating the cytotoxicity of Fusarium mycotoxins. The replacement of MTT by MTS substantially reduced the number of sample processing steps and the length of time required to complete the cytotoxicity assay.

Optimization of the L-M cell bioassay for quantitating tumor necrosis factor α in serum and plasma

In attempting to quantitate TNF-α in body fluids using current bioassay protocols, we discovered several factors which adversely affect reliability, sensitivity and specificity. The objective of this study was to establish an optimum assay for use with serum and plasma samples. While adopting the commonly used L-M cell bioassay to test serum and plasma samples, we noted non-specific staining of cell debris and protein by crystal violet dye, even if the wells were washed prior to staining. However, when we replaced crystal violet with tetrazolium salts to measure cell viability, we discovered that both serum and plasma, from a variety of species, non-specifically reduced both MTT and XTT tetrazolium salts to a colored formazan product resulting in absorbance values significantly higher than those of medium and serum controls. This effect was particularly pronounced with fetal bovine serum which showed significant color development with as little as 6% serum in the test wells. Maximum sensitivity can only be obtained by eliminating these false negative readings. Therefore the serum or plasma containing supernatant must be removed from the test wells and replaced with fresh serum-free medium prior to addition of the substrate. This finding should be applicable to other body fluids such as urine, milk, and synovial fluid as well as any tetrazolium based assay for cell viability which uses serum-supplemented culture medium. Additionally, when the substrate XTT, which reduces to a water-soluble formazan product, was compared to MTT which reduces to a water-insoluble product, XTT was found to be superior since elimination of the solubilization process resulted in reduction of assay time. Also, overall absorbance readings using XTT were higher than with MTT, without any loss of sensitivity. There were no differences in detectability of TNF-α between serum and plasma and use of preservative-free heparin was found not to have adverse effects on TNF-α assay. Heat treatment of both serum and plasma seemed to inactivate factors that contributed to the non-specific lysis of the L-M cells when their concentrations exceeded 25%.

Comparative analysis of using MTT and XTT in colorimetric assays for quantitating bovine neutrophil bactericidal activity

Two different tetrazolium compounds were compared for use in a colorimetric assay for quantitating bovine neutrophil bactericidal activity against Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, and Brucella abortus. The tetrazolium compounds tested included 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and sodium 3,3′-[1(phenylamino)carbonyl]-3,4-tetrazolium]-3is(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT). The MTT and XTT colorimetric bactericidal assays were conducted by incubating antibody-opsonized bacteria with neutrophils in microtiter plates for 30 and 60 min at ratios of ten and 100 bacteria per neutrophil. Neutrophils were then lysed with saponin and samples were incubated 30 min with MTT or XTT plus coenzyme Q (CQ). Dead bacteria and lysed neutrophils did not react with MTT or XTT plus CQ. Live bacteria converted XTT to water soluble orange formazan in the presence of CQ and MTT to insoluble purple formazan. Absorption of formazan produced by bacteria from XTT was measured at 450 nm. Formazan produced by bacteria from MTT was solubilized by adding isopropanol and measured by absorption at 560 nm. Absorption of both types of formazan was directly related to viable bacteria cell number and used to determine the number of bacteria not killed by neutrophils. The percentage of bacteria killed by neutrophils was determined by extrapolation from a standard formazan curve that was derived by incubating MTT or XTT plus CQ with known numbers of bacteria. The XTT and MTT colorimetric bactericidal assays produced comparable results when used to measure bovine neutrophil bactericidal activity against S. aureus, E. coli, L. monocytogenes, and B. abortus. However, the assay using XTT was quicker and easier to perform because bacteria converted XTT to a formazan that did not need to be solubilized before measuring absorption.

A New Sulfonated Tetrazolium Salt That Produces a Highly Water-Soluble Formazan Dye.

A new tetrazolium compound, sodium salt of 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate (1a), which produces a highly water-soluble formazan dye, due to the presence of two sulfonate groups, was synthesized and its potential utility evaluated in assays of NADH and cell proliferation. The compound proved to have a similar sensitivity to 2, 3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) in the assay of NADH and was also useful as an indicator of cell viability, with less cytotoxicity than XTT, in the proliferation assay using P388 cell lines.