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PubMed1987

Some acute effects of monochromatic ultraviolet B irradiation on mouse epidermis measured by the tetrazolium-reduction test and determination of DT-diaphorase activity, with reference to carcinogenesis.

Some acute epidermal effects of monochromatic ultraviolet B (UVB) irradiation on hairless mouse skin were measured by the tetrazolium test (TZT), and by determining the DT-diaphorase activity in epidermal cells. Dose response and time course studies were carried out after UVB irradiation at 280, 290, 297 and 302 nm. Appropriate UV doses at all the wavelengths increased the cellular deposition of formazan (TZT). At higher doses the epidermal cells became too injured to react. Wavelengths at 280 and 290 nm seemed more injurious than those at 297 and 302 nm. There was, however, no increase in DT-diaphorase activity after UVB irradiation. This indicates that the increased formazan deposition (TZT) after UVB is more likely to be caused mainly by membrane effects. Detoxification mechanisms which activate DT-diaphorase, as often seen after cellular contact with chemical carcinogens, are not involved.

Animals↗

PubMed1991

A quantitative microwell assay for chondrocyte cell adhesion.

An assay for measuring the quantity of live articular chondrocytes attached to a substratum in microwell plates was established by measuring the absorbance of the blue formazan product generated from the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-dypenyl tetrazolium bromide (MTT). Blue formazan production was optimal at an MTT concentration of 1 mg/ml (100 microliters per microwell) and an incubation period of 3 h. The absorbance of the dye was linearly related to the quantity of cells added per microwell. The number of live chondrocytes attached to adhesive proteins can be quantitated using this technique.

Animals↗

PubMed1995

Localization of NADPH-diaphorase/nitric oxide synthase in the rat retina: an electron microscopic study.

The activity of NADPH-diaphorase (NADPH-d), a marker for nitric oxide synthase (NOS), was examined histochemically in the rat and mice retina. Mice in which the neuronal NOS gene has been disrupted (nNOS- mice) were used for specificity controls. Light microscopically a few amacrine cells were heavily stained. Other cells were stained weakly or not at all. Under the electron microscope, formazan precipitates were detectable on membranes of endoplasmic reticulum, nuclear envelope, mitochondria, and, in a few cases, the Golgi complex. Bipolar, horizontal, and Müller cells, were if at all, sparsely labeled with formazan. Labeled mitochondria were observed in rod endings and in inner segments of photoreceptors. Outer segments of photoreceptors and ganglion cells were completely free of reaction product. The NADPH-d reaction in wild-type mice displayed a similar distribution pattern to that in rats. Retinae of nNOS- mice showed a complete lack of prominent NADPH-d stained (amacrine) cells. None or a very few labeled membranes were seen.

Animals↗

PubMed1987

The quantitation of HLA-DR expression on human cells using immunocytochemistry.

An immunocytological method that can be used to quantify the comparative expression of Class II HLA-DR antigens on human cells has been developed. Tissue sections or cytospins are incubated with the monoclonal antibody RFDR1 (anti-HLA-DR), conjugated to the enzyme glucose oxidase (GO). The bound McAb is identified by incubation with the substrate beta-D-glucose and capture of the reducing equivalents generated by a tetrazolium salt forming an insoluble formazan. Under standard conditions the amount of formazan precipitated is proportional to the amount of McAb bound to the cells or tissue and thus the amount of HLA-DR expressed. The reaction has been quantified both by spectrophotometry (on tissue sections) and by microdensitometry (on cell spreads). Computer analysis has been used to relate density of HLA-DR expression to cell area. Repeated 'blind' measurements by separate investigators have confirmed the reproducibility of the results. It is suggested that this method adds a new dimension to immunohistological/cytological analysis of tissues and single cells.

Antibodies, Monoclonal↗

PubMed1989

Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill.

The tetrazolium salt (MTT) method involving conversion of MTT to coloured formazan by cells serving as indirect measurements of cell growth/cell kill has been reported by several groups, although technical problems have been encountered. The present investigation was undertaken in order to delineate what laboratory variables have direct influence on the sensitivity and reproducibility of the method. The pH of the extraction buffer was of the utmost importance, since it was demonstrated that a pH greater than 5 would give rise to false signals. Furthermore, modifying the composition of the extraction buffer, all formazan dye grains were solubilised, totally. A direct comparison with published methods demonstrated that only the modified method would yield 100% higher signals without increasing the background. In contrast to previous reports, it was shown that phenol red does not interfere with the measurements and no washing steps are required since all ingredients can be added subsequently. Serum proteins at concentrations up to 25% have no influence on the result. All samples can be measured in an ELISA scanner at 570 nm with little intra-assay variation.

Animals↗

PubMed1988

Use of the MTT assay for rapid determination of chemosensitivity of human leukemic blast cells.

A microcytotoxicity assay employing a tetrazolium salt has been adapted for testing the response of human leukemic blast cells to a variety of chemotherapeutic agents. After exposure to various concentrations of drugs, the viability of fresh leukemic blast cells was measured using a tetrazolium salt, MTT, which is converted to blue formazan crystals by living cells. The amount of formazan produced was quantitated using a microtitre plate spectrophotometer. In the present study, optimal conditions for chemosensitivity testing of human leukemia samples were determined, and the relative chemosensitivity of five patient samples was tested.

Antineoplastic Agents↗

PubMedDate not supplied

Determination of potassium/sodium ratio in heart tissue. Evaluation of its use as an index of myocardial ischaemic damage. Comparison with the nitro-BT test.

The succinate dehydrogenase method of staining and the determination of the K+/Na+ ratio of heart tissue have been applied to forensic material as a tool for demonstrating early signs of myocardial infarctions. Both methods are controlled by a series of forensic cases dying from causes other than arteriosclerosis of the coronary arteries. Based on an analysis of normal material some factors influencing the K+/Na+ ratio are defined. It is concluded that the determination of the K+/na+ ratio shows more infarcts than the formazan method; however, the evaluation of the K+/Na+ ratio is difficult. The K+/Na+ determination is not recommended for routine use. A possible indication for the use of the K+/Na+ determination together with the formazan test could be cases of instantaneous death by accidents suspected to have been provoked by a fatal heart attack.

Adolescent↗

PubMed1993

Genistein inhibits tumour cell growth in vitro but enhances mitochondrial reduction of tetrazolium salts: a further pitfall in the use of the MTT assay for evaluating cell growth and survival.

The natural isoflavone genistein inhibits the growth of a number of tumour cell lines in vitro. During investigations on the antiproliferative effects of genistein we observed that, with respect to direct cell counting, a tetrazolium (MTT) colorimetric assay consistently underestimated the growth inhibitory activity of the substance. Cell proliferation was markedly inhibited by genistein in three tumour cell lines (MCF-7, human breast tumour; Jurkat cells, human T-cell leukaemia; L-929, mouse transformed fibroblasts) when cell number was evaluated by direct counting, whereas a 72-h MTT assay failed to reveal any growth-inhibitory effect. Cell cycle analysis by propidium iodide staining and flow-cytometry revealed a G2/M cell cycle arrest after genistein treatment. Genistein-treated cells displayed an increase in cell volume and in mitochondrial number and/or activity, as revealed by enhanced formazan generation and increased uptake of the vital mitochondrial dye rhodamine 123. These results suggest that alterations in cell cycle phase redistribution of tumour cells by genistein may significantly influence mitochondrial number and/or function and, consequently, MTT reduction to formazan. This may constitute an important bias in analysing the effects of genistein, and possibly other drugs that block the G2/M transition, on growth and viability of cancer cells in vitro by MTT assay.

Animals↗

PubMed2005

A novel and rapid method to quantify cytolytic replication of picornaviruses in cell culture.

Determining viral titers is a key issue in a wide variety of studies regarding different aspects of virology. The standard methods used for determining picornavirus titers are endpoint titration assay and plaque assay, both time consuming and laborious. The method described uses the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide) that is reduced to formazane by cellular dehydrogenase, genes shown to be down-regulated during picornavirus infection. The amount formazane produced correlates with the viral titers obtained and can easily be measured using an ELISA plate reader. The colorimetric method has been evaluated using virus types from different genera of the Picornaviridae family. The MTT method reduces the time spent on determining the viral titers and still maintains a reliable accuracy.

Animals↗

PubMed1994

On the mechanism of the multistep reduction of tetrazolium salts with special reference to the involvement of tetrazolium radicals.

Thin layer chromatography (TLC) was performed to analyze which products are formed when unstable tetrazolinyl radicals are generated during the reduction of tetrazolium salts under physiological conditions of biochemical and histochemical assays. It was found in aqueous media that irrespective the assay conditions, reduction of 2,3-di(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (p-DNTTC) or 2,3-di(4-nitrophenyl)-5-t-butyl-2H-tetrazolium chloride (t-butyl-DNTC) always lead to production of formazan and never to generation of the corresponding 2(4-nitrophenyl)-5-phenyl- (respectively t-butyl-) tetrazoles by splitting off a nitrophenyl side chain from the tetrazolium ring as a cation. This reaction is known to occur in non-aqueous media (Neugebauer, 1973). Because p-DNTTC is analogous to tetrazolium salts that are used in biochemical and histochemical assays, it is concluded that reduction of tetrazolium salts leads to formation of formazans only under these conditions.

Chromatography, Thin Layer↗

PubMed1995

A novel quantitative histochemical assay to measure endogenous substrate concentrations in tissue sections. Fundamental aspects.

A quantitative histochemical assay has been developed for measurement of endogenous substrate concentrations in cryostat sections using a colorimetric visualization technique. Model sections of frozen gelatin solutions with known concentrations of glucose-6-phosphate (G6P) were sandwiched with a second cryostat section containing glucose-6-phosphate dehydrogenase (G6PDH) and all other compounds (with the exception of G6P) that are necessary for the demonstration of G6PDH activity with a tetrazolium salt method. After 60 min of incubation, G6P was converted with concomitant formazan production. The amount of formazan generated was measured cytophotometrically and used as a parameter of the G6P concentration in the first section. A calibration graph was obtained with a high correlation coefficient, allowing the conversion of mean integrated absorbance values into absolute substrate concentrations. The method was highly reproducible, and the recovery of G6P was 85 +/- 4% irrespective section thickness (4-20 microns) and G6P concentration (0.08-1.6 mM) in the sections. The sensitivity of the tetrazolium-linked method appeared to be 100 microM in 20 microns thick sections. This sensitivity enables the measurement of physiological substrate concentrations in tissue sections. Spatial resolution was approximately 150 microns, indicating a relatively high rate of diffusion of G6P during the reaction. The model study shows that the method described here allows the quantitative determination of substrate concentrations in tissue sections. These endogenous substrate concentrations are necessary for the calculation of local metabolic fluxes when determined in combination with local enzyme activities and kinetics, thus giving a more accurate reflection of in situ metabolic heterogeneity of tissues.

Calibration↗

PubMed2003

A rapid MTT colorimetric assay to assess the proliferative index of two Indian strains of Theileria annulata.

A study was undertaken to compare the proliferative index of macroschizont-infected lymphoblastoid cells of two Indian strains [Izatnagar (IZT) and Parbhani (PBN)] of Theileria annulata by an in vitro MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide], colorimetric assay. Culture conditions were standardized to define the optimal cell concentration in 96-well microculture plates to yield nearly 100% living cells for measurement of the metabolized formazan activity. A cell concentration of 1.5x10(5) cells/ml was found to be optimal for effective discrimination of the parasite strains. On the basis of conversion of MTT by the actively proliferating lymphoblastoid cells, the PBN strain of T. annulata stimulated a 2.5-fold increase in formazan activity in comparison to the IZT strain. The in vitro MTT assay was found to be a simple and convenient method for assessing the cell activation rate and growth, obviating the need for radioactive material for the assay. The results of the proliferation assay are discussed in relation to previously documented information on the biological characteristics of this important pathogen of cattle.

Animals↗

PubMed2000

Mitotic viability and metabolic competence in UV-irradiated yeast cells.

Colony formation is the classic method for measuring survival of yeast cells. This method measures mitotic viability and can underestimate the fraction of cells capable of carrying out other DNA processing events. Here, we report an alternative method, based on cell metabolism, to determine the fraction of surviving cells after ultraviolet (UV) irradiation. The reduction of 2,3,5-triphenyl tetrazolium chloride (or TTC) to formazan in mitochondria was compared with cell colony formation and DNA repair capacity in wt cells and two repair-deficient strains (rad1Delta and rad7Delta). Both TTC reduction and cell colony formation gave a linear response with different ratios of mitotically viable cells and heat-inactivated cells. However, monitoring the formation of formazan in non-dividing yeast cells that are partially (rad7Delta) or totally (wt) proficient at DNA repair is a more accurate measure of cell survival after UV irradiation. Before repair of UV photoproducts (cis-syn cyclobutane pyrimidine dimers or CPDs) is complete, these two assays give very different results, implying that many damaged cells are metabolically competent but cannot replicate. For example, only 25% of the rad7Delta cells are mitotically viable after a UV dose of 12 J/m(2)75% of these cells are metabolically competent and remove over 55% of the CPDs from their genomic DNA. Moreover, repair of CPDs in wt cells dramatically decreases after the first few hours of liquid holding (L.H.; incubation in water) and correlates with a substantial decrease in cell metabolism over the same time period. In contrast, cell colony formation may be the more accurate indicator of cell survival after UV irradiation of rad1Delta cells (i.e., cells with little DNA repair activity). These results indicate that the metabolic competence of UV-irradiated, non-dividing yeast cells is a much better indicator of cell survival than mitotic viability in partially (or totally) repair proficient yeast cultures.

Colony Count, Microbial↗

PubMed2003

A new method for studying the adhesion of Candida albicans to dentin in the presence or absence of smear layer.

OBJECTIVES: The purpose of this study was to develop a reproducible, quantitative model of Candida albicans adhesion to human dentin through the use of a colorimetric method and to evaluate the effect of smear layer on candidal adhesion. STUDY DESIGN: Dentin disks with or without smear layer were incubated with C albicans (10(8) cells/mL) for 4 hours. After incubation, the disks were exposed to an (2,3)-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium hydroxide-coenzyme Q solution for 2 hours. The color of (2,3)-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium hydroxide formazan in the supernatant was determined spectrophotometrically at 492 nm. To relate formazan formation to cell numbers, standard curves were generated with known numbers of yeast cells without dentin. The number of adherent cells per square millimeter was then calculated. RESULTS: The number of attached C albicans cells was 2.4 x 10(4) per square millimeter in dentin with smear layer and 1.5 x 10(4) in dentin without smear layer (P <.05). CONCLUSION: (2,3)-Bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium hydroxide assay is a potential microbiologic tool for the quantitative determination of Candida adhesion to human dentin.

Candida albicans↗

PubMed2003

A colorimetric assay for determination of cell viability in algal cultures.

In this work, we propose the determination of cell viability in algal cultures by using a colorimetric assay widely used for estimation of cell proliferation in animal cell cultures. The method is based on in vivo reduction by metabolically active cells of a tetrazolium compound (MTS=3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium, inner salt) to a colored formazan, with maximal absorbance at 490 nm, that is released to the culture medium. For this purpose, we have tested two microalgae with high commercial value (Dunaliella and Spirulina) and two seaweeds with different morphology (Ulva and Gracilaria). Color development in this assay is directly proportional to the number of viable cells, to the incubation time in the presence of the assay solution, and to the incubation temperature. A direct significant correlation was found between algal photosynthesis rate and color development in all species used through this work. Moreover, the intensity of absorbance at 490 nm was significantly lower in stressed cells (e.g. in nutrient-limited cultures, in the presence of toxic substances, and in osmotically-stressed cultures). We conclude that cell viability of algal cultures can be rapidly and easily estimated through colorimetric determination of the reduction of MTS to formazan.

Adaptation, Physiological↗

PubMed2003

Inhibition of oxygen radical formation by methylene blue, aspirin, or alpha-lipoic acid, prevents bacterial-lipopolysaccharide-induced fever.

Phagocytic cells contain NADPH oxidase that they use for host defense by catalyzing the production of superoxide. Bacterial lipopolysaccharide (LPS) has been found to stimulate NADPH oxidase in mobile and sessile macrophages and microglia. It also evokes fever in homeothermic animals and men, a reaction mediated by central nervous system (CNS) activities. The purpose of the present study was to determine whether reactive oxygen species are involved in LPS-induced fever. In rabbits we found that plasma hydroperoxide levels increased and catalase activity decreased 15 min after LPS injection and that fever started with a similar latency, while plasma levels of tumor necrosis factor-alpha (TNFalpha) increased 30 min after the injection. Treating rabbits with methylene blue or aspirin did not affect TNFalpha secretion but prevented the LPS-induced rise of hydroperoxides and the inactivation of catalase, abolishing fever. Incubation of human blood with nitroblue tetrazolium and LPS increased the number of formazan-positive neutrophils from 10 +/- 5 to 52 +/- 9%. Adding LPS to blood preincubated with either methylene blue, alpha-lipoic acid, or aspirin respectively decreased the number of formazan-positive neutrophils to 0.9 +/- 0.8, 0.8 +/- 0.9, or 2.0 +/- 0.9%, disclosing the antioxidant capacity of these drugs. Systemic application of 80 mg/kg alpha-lipoic acid elicited heat-loss reactions within 15 min and decreased core temperature by 2.2 +/- 0.3 degrees C within 2 h. Alpha-lipoic acid applied 45 min after LPS induced antipyresis within 15 min, and this antipyresis was associated with a decrease of elevated hydroperoxide levels and restoration of catalase activity. Our results show that fever is prevented when the production of reactive oxygen species is blocked and that an elevated body temperature returns to normal when oxygen radical production decreases. Estimation of plasma dihydrolipoic acid (DHLA) levels following injection of 80 mg/kg alpha-lipoic acid in afebrile and febrile rabbits revealed that this acid is converted into DHLA, which in afebrile rabbits increased the plasma DHLA concentration from 2.22 +/- 0.26 microg/ml to peak values of 8.60 +/- 2.28 microg/ml DHLA within 30 min and which in febrile rabbits increased it from 0.84 +/- 0.22 microg/ml to peak values of 3.90 +/- 0.94 microg/ml within 15 min. Methylene blue, aspirin, and alpha-lipoic acid, which all cross the blood-brain barrier, seem to act not only on peripheral tissues but also on the CNS. Brain structures that have been shown to sense oxidative stress are vicinal thiol groups attached to the NMDA subtype of glutamate receptor. Their reduction by thiol-reducing drugs like dithiothreitol or DHLA has been found to increase glutamate-mediated neuronal excitability, while the opposite effect has been observed after their oxidation. Because we found that systemic application of alpha-lipoic acid in the afebrile state elicits hypothermia and in the febrile state is antipyretic, we think this type of NMDA receptor is involved in thermoregulation and that oxidation of its thiol groups induces fever. It appears that temperature homeostasis can be maintained only if the redox homeostasis of the brain is guaranteed.

Animals↗

PubMed1989

The MTT assay underestimates the growth inhibitory effects of interferons.

The growth inhibitory effects of interferons, IFN-alpha and IFN-gamma on human lung cancer cell lines were studied using both a tetrazolium (MTT) colorimetric assay and direct cell counting. Significant discrepancies between the two assays were observed, the MTT assay consistently underestimating the growth inhibitory effects of the IFNs. There was no direct chemical effect of the IFNs on the tetrazolium reduction process. IFN treated cells showed increased cell size compared with control cells, although there was little or no change in cell cycle distribution. Mitochondrial activity was 30-50% greater in IFN-gamma treated cells (COR-L23) than the controls. Reduced formazan production per cell was observed in medium which had supported cell growth for several days. Differential 'medium conditioning' led to a difference in formazan production per cell between IFN and control cells and this was the major basis of the observed discrepancy. This discrepancy was not due to the differences in the glucose concentrations between these media. However, differences in pH between the media proved to be the major contributory factor of the discrepancy.

Cell Count↗

PubMed1998

Interference with cellular energy metabolism reduces kynurenic acid formation in rat brain slices: reversal by lactate and pyruvate.

This study was designed to investigate the role of cellular energy metabolism in the de novo formation of the endogenous excitatory amino acid receptor antagonist, kynurenic acid. Using rat cortical tissue slices, the roles of glucose transport, glycolysis, tricarboxylic acid cycle intermediates and oxidative phosphorylation were studied. Inhibition of glucose utilization resulted in quantitatively similar decreases in kynurenine uptake, kynurenic acid production and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, a marker of mitochondrial activity. The end product of glycolysis, pyruvate, as well as lactate, attenuated all three deficits. Pyruvate also significantly increased kynurenic acid formation in normal brain slices without affecting kynurenine uptake. Oxaloacetate and alpha-ketoglutarate (tricarboxylic acid cycle intermediates) were the only compounds tested which were capable of duplicating the effects of pyruvate, indicating that 2-oxoacids can stimulate kynurenic acid synthesis by acting as aminoacceptors in the enzymatic transamination of kynurenine. When the mitochondrial electron transport chain was blocked by specific inhibitors, coincubation with succinate restored the rate of MTT formazan formation to normal (except in the case of 3-nitropropionic acid), yet failed to prevent the resulting reduction in kynurenic acid synthesis. Conversely, pyruvate increased kynurenic acid production in the presence of all inhibitors (except cyanide), but did not attenuate the reduction in kynurenine uptake and MTT formazan formation. Taken together, these results demonstrate that interference with cellular energy metabolism causes mechanistically diverse, pronounced reductions in the cerebral neosynthesis of kynurenic acid, and that 2-oxoacids and lactate can effectively reverse most of these detrimental effects.

Animals↗