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At least 235 records · Page 13Linked to original sources
PubMed2002

[Novel quantitative method for evaluating oxygen/glucose deprivation-induced injury of hippocampal slices]

OBJECTIVE: To establish a simple, sensitive in vitro method to evaluate oxygen/glucose deprivation (OGD)-induced injury of brain hippocampal slices in rats. METHODS: Rat hippocampal slices were incubated in 2% 2, 3, 5-triphenyltetrazolium chloride (TTC) solution after oxygen/glucose deprivation. They were then soaked in a measured volume of ethanol and dimethylsulfoxide (50:50) to extract the TTC formazan Product which was then measured by spectrophotometry. OGD induced LDH release was simultaneously measured. RESULTS: Progressive prolongation of OGD induced hippocampal injury resulted in decreased formazan coloration as determined by spectrophotometry. There was a parallel increase in LDH release, thus a negative correlation in these two products was noted. (r=-0.933,P <0.01). The injury was attenuated in the brain slices pre-treated with nimodipine, dexamethasone, and ketamine, but not ONO-1078. CONCLUSION: Solvent extraction and spectrophotometric quantification of formazan represents an objective measurement of OGD-induced injury of rat hippocampal slices.

Journal Article↗
PubMed2000

Antioxidant compounds interfere with the 3.

Antioxidants are often added to culture media as cytoprotective agents. We examined the effects of antioxidants on the results and interpretation of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay for cell viability. Without cells, the thiol-containing antioxidant compounds beta-mercaptoethanol, dithiothreitol, pyrrolidine-dithiocarbamate, and N-acetyl-L-cysteine (acetylcysteine) reduced MTT tetrazolium salts to a blue formazan product in a dose-dependent manner. Addition of the compounds L-ascorbic acid and (+)-alpha-tocopherol acid succinate had different effects. In contrast, addition of the antioxidants N-acetyl-5-methoxytryptamine and (-)-2-oxo-4-thiazolidine carboxylic acid, which do not contain reactive thiol groups, did not result in the development of blue formazan product. These results showed that antioxidants, and potentially other chemotherapeutic compounds that contain free thiol groups or other reducing equivalents, readily reduce MTT to produce the blue formazan, irrespective of the viability of the cells present. This undescribed reaction can, therefore, significantly influence the results and interpretation of cell-viability experiments.

Acetylcysteine↗
PubMed1992

Cytoprotective effect of dilazep on hydrogen peroxide-perturbed vascular endothelial cells.

The effect of dilazep and dimethyl thiourea (DMTU) on the hydrogen peroxide-derived injury of culture pulmonary artery epithelial cells (CPAEC) was assessed by colorimetric assay of MTT formazan (MTT formazan assay). When CPAEC were treated with hydrogen peroxide, neither cell lysis nor detachment of the cells from surface of the well was observed. However, the MTT formazan formation was decreased in a time and dose dependent manner. The decrease in the formation was significantly suppressed in the presence of dilazep (0.1 to 10 microM) or DMTU (0.01 to 0.3 microM). CPAEC treated with hydrogen peroxide in the same way enhanced an activation of prothrombin, and this enhancement was significantly inhibited in the presence of dilazep (1 to 3 microM). These data indicate that dilazep exerts a cytoprotective effect against challenges of intracellular oxidant produced by hydrogen peroxide and suppresses augmented procoagulant activity of injured cells.

Animals↗
PubMedDate not supplied

[Photodesorption of mitochondria].

A photodesorption of mitochondria absorbed on a quartz plate was found. The rate of desorption depends on the wavelength, and the intensity and time of irradiation. The maximal rate of photodesorption was detected upon ultraviolet irradiation at the absorption band of mitochondrial proteins. Probably, the photodesorption is caused by a local photothermal effect: a heating of photoexcited surface-membrane proteins, which attach mitochondria to the quartz plate. Preliminary fixation of a smear by isopropanol preserves the spontaneous desorption. No photodesorption of either mitohondria or formazan was observed upon irradiation of the smear with formazan by visible light (wavelength 540 nm; formazan was formed in the NADH-pNTV:reductase reaction). The data obtained are important for the elaboration of technology of mitochondrial immobilization in measurements of the enzyme activity and for biocensors.

Adsorption↗
PubMed1991

The effects of ivermectin on the viability of Onchocerca lienalis microfilariae in vitro and on their subsequent development in the blackfly vector, Simulium ornatum.

Experiments examined the sensitivity of Onchocerca lienalis microfilariae (mf) to ivermectin in vitro using a range of parameters to measure viability. Incubation in drug (1 x 10(-4) and 5.71 x 10(-8) M) caused an immediate decrease in motility levels in a concentration dependent fashion with a continuing decline over 24 h; the inclusion of a monkey kidney cell (LLCMK2) feeder layer in the culture system partially abrogated drug effects. The use of feeder cells allowed the longer-term culture of mf; incubation in drug at low concentrations (5.71 x 10(-8) and 1 x 10(-10) M) significantly reduced motility levels during a 17 day trial. Using MTT colorimetry it was found that 10,000 untreated mf per replicate sample were required to produce a sufficient quantity of formazan to make comparisons, and that formazan formation was most rapid during the first 30 min of incubation in MTT but continued up to 3-4 h. Following incubation of mf in ivermectin (1 x 10(-4) and 5.71 x 10(-8) M) for 24 h, only the higher concentration inhibited formazan formation (51%), while motility levels were reduced by 94% and 45% respectively. Using the same drug concentrations, mf viability was assessed by measuring the uptake of [3H]2-deoxy-D-glucose; uptake was only significantly reduced (40%) by the higher concentration, while motility levels were reduced by 77% and 63% respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1981

Some aspects of the use of tetrazolium salts in quantitative histochemistry.

The reduction of tetrazolium salts to highly coloured insoluble formazans is a reaction which has been widely exploited in histochemistry. This presentation considers three important aspects of the use of these compounds in quantitative studies, namely purity, intermediate reduction products, and calibration and measurement. Thin layer chromatography of tetrazolium salts from different suppliers has shown that standards of purity can vary widely, and that some samples are seriously contaminated. Studies with chromatographically pure compounds have demonstrated that the red diffuse colour seen in some reactions with di-tetrazolium salts is due to a half-reduced reduction intermediate. This "half-formazan" is part of the total activity, and cannot be disregarded in quantitative work. An understanding of the chemical nature of formazan end-products in tissue sections has made it possible to calibrate these reactions for microdensitometry.

Chromatography, Thin Layer↗
PubMed1977

[Nitroblue tetrazolium test in Hodgkin's disease (author's transl)].

Nitroblue tetrazolium dye (NBT) test was performed in 60 patients suffering from Hodgkin's disease. The control group consisted of 60 healthy adults. 23 patients with inactive lymphogranulomatosis had a mean NBT-balue of 30 +/- 9% formazan cells. There was no difference compared with controls (32 +/- 10% formazan cells). 37 patients suffered from active Hodgkin's disease. In this group 31 patients showed an elevated value, 6 had normal levels. On average the level was significantly raised with 59 +/- 20% formazan cells (p less than 0,01). A correlation between the extent of the disease and the height of the NBT figure could not be demonstrated. The same applies to the different histological types. In comparison between the NBT value and the simultaneously examined ALP it was found that ALP is a more sensitive parameter but that an increased ALP is not indicative of an active stage. It is therefore advisable to determined both parameters to get more security whether there is active or inactive Hodgkin's disease.

Alkaline Phosphatase↗
PubMedDate not supplied

Semiautomated quantification of cytotoxic damage induced in cultured insect cells exposed to commercial Bacillus thuringiensis biopesticides.

A convenient in vitro bioassay based on semiautomated quantification of live-cell reduction of tetrazolium dyes--3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-corb oxanilide sodium salt (XTT)--to formazan was developed and used to evaluate cytotoxic effects of two commercial insecticides (BT) derived from Bacillus thuringiensis subsp. kurstaki (Btk). Comparison of two target insect cell lines MG1 (Trichoplusia ni, cabbage looper midgut) and Sf9 (Spodoptera frugiperda, fall army worm oocyte) revealed similar cell-dependent responses in mitochondrial-associated electron transport activity. The 50% inhibition of formazan production (ID50) obtained by exposing these cells to 20 microM 2,4-dinitrophenol or 5 mM sodium azide occurred in the range 10(-7)-10(-6) International Units (IU) of BT cell(-1) 24 h(-1). Damage to cell adhesion and cytoarchitecture, revealed by light and electron microscopic analysis, increased with BT exposure and dose. MTT was superior to XTT as a cytotoxic indicator in kinetic studies related to spores, a major component of BT. Unless blocked by antibiotic (gentamicin), vegetative growth resulting from spore germination was the major cause of toxicity. The ID50 exposure time using vegetative Btk cells was approximately 0.1-0.2 times that required for BT spores, with or without intact parasporal proteins present. This difference in exposure is an indirect measure of the time required for spores to germinate and produce vegetative cells. The assay methodology developed here, if linked with suitable target and non-target animal cell types, should have broad application for conducting standardizable estimates of cytotoxic potential of any microbe-based biotechnology product.

Bacillus thuringiensis↗
PubMed1989

In situ evaluation of the stimulatory state of hepatic macrophages based on their ability to produce superoxide anions in rats.

When a liver perfusion with nitro blue tetrazolium (NBT) and phorbol myristate acetate (PMA) was performed in carbon tetrachloride (CCl4)-intoxicated rats, formazan deposition was remarkable in macrophages in the necrotic areas of the liver, its intensity varying with the extent of injury. The deposits almost disappeared after addition of Cu(Lys)2, a scavenger of intra- and extracellular superoxide, but were not affected by superoxide dismutase (SOD), which acts extracellularly. The formazan content after incubation with NBT and PMA was higher in macrophages isolated from CCl4-intoxicated liver than in those from normal liver, though their PMA-induced chemiluminescence did not differ. In Corynebacterium parvum-treated liver, both Cu(Lys)2 and SOD reduced the deposits. This method can estimate in situ the ability of hepatic macrophages to produce superoxide and the cellular sites of its production.

Animals↗
PubMed1995

The effect of detergents on the reduction of tetrazolium salts.

Detergents, such as Triton X-100, markedly increase the reduction of tetrazolium salts by xanthine oxidase plus xanthine, or by NADH. This effect of detergent, in the case of the xanthine oxidase catalyzed process, is seen aerobically but not anaerobically. Increasing the rate of accumulation of formazan, whether by increasing the concentration of the tetrazolium salt or by adding detergent, decreased susceptibility to inhibition by superoxide dismutase or by O2. These results are accommodated by a scheme of reactions the essence of which is the univalent reduction of the tetrazolium to an uncharged tetrazoinyl radical which can reduce O2 to O2- or which can partition into the detergent micelles and there dismute to generate the stable formazan.

Detergents↗
PubMed2000

The role of plasma membrane in bioreduction of two tetrazolium salts, MTT, and CTC.

Despite widespread use of various tetrazolium assays, the mechanisms of bioreduction of these compounds have not been fully elucidated. We investigated the capacity of tetrazolium salts to penetrate through intact cell plasma membranes. 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) tetrazolium salts appear to represent examples of species that are reduced by different mechanisms. We provide evidence suggesting that MTT readily crosses intact plasma membranes and is reduced intracellularly. MTT appears to be reduced by both plasma membrane and intracellular reductases; reducing cells are not damaged and remain metabolically active for at least 45 min. In contrast, CTC remains extracellular with respect to viable cells and thus requires plasma membrane permeable electron carrier to be reduced efficiently. However, reduction of CTC in the presence of an electron carrier inflicts damage on plasma membranes. The intracellular vs extracellular sites of reduction of tetrazolium salts were established on the basis of deposition of formazans. Crystals of formazan were detected using fluorescence or backscattered light confocal laser microscopy. We postulate that the capacity of a tetrazolium salt to cross intact plasma membranes constitutes an important experimental variable which needs to be controlled in order to correctly interpret the outcome of tetrazolium assays designed to measure cellular production of oxygen radicals, activity of mitochondrial, cytosolic, or outer membrane reductases, etc.

Biological Transport↗
PubMed1997

Cycling assay for nicotinamide adenine dinucleotides: NaCl precipitation and ethanol solubilization of the reduced tetrazolium.

The spectrophotometric cycling assay for the determination of nicotinamide adenine dinucleotides involving 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide as the final electron acceptor has been improved. The enzyme reaction is stopped by the addition of high concentrated NaCl, which accelerates the precipitation of the formazan. After isolation from the assay medium, the formazan is easily solubilized in ethanol. This allows more flexibility and sensitivity, offering additionally the possibility to reduce the amount of reagents required. This method was applied for extracts of green tissues of higher plants, and the results did not differ from those obtained with the original procedure.

Brassica↗
PubMed1975

Histochemical study of monoamine oxidase latency in the liver of the rat.

In a histochemical test system with adrenaline as substrate and nitroblue tetrazolium (NBT) as electron acceptor, an increase of NBT reduction in rat liver sections was found microspectrophotometrically following short hypotonic treatment. Investigations with iproniazide, a monoamine oxidase inhibitor, and non-enzymatic NBT reduction showed that the increased formazan formation was related to the presence of monoamine oxidase. It is suggested that the reason for the observed increase of formazan formation is due to increased permeability of the inner mitochondrial membrane to NBT. Consequently, the increase of monoamine oxidase observed in the histochemical test system does not represent mobilization of a latent activity, but rather complete assessment of activity that is normally present.

Animals↗
PubMed1979

Quantitative succinate-dehydrogenase histochemistry. I. A Methodological study on mammalian and fish muscle.

In enzyme histochemistry formazan production can be used as a measure for oxidative enzyme activity. The formazan deposits can be measured quantitatively per cell with a scanning and integrating microspectrophotometer. Optimal conditions are described for the estimation of histochemical succinate dehydrogenase activity in sections of fish bodymusculature and mouse soleus and plantaris muscle. It is shown that when proper measuring conditions are chosen a ditetrazolium salt (TNBT) can be used in quantitative enzyme histochemistry and that the optimal conditions for the histochemical succinate dehydrogenase reaction in muscle fibres of fish and mouse muscle are somewhat different for these two species. The differences in pH, temperature and succinate sensitivity are the most prominent.

Animals↗
PubMed1978

Quantitation of mitochondrial injury in cultured rat heart endothelioid cells with nitroblue tetrazolium.

A sensitive method is presented for measurement of changes in the permeability of mitchondria in cultured cells. Rat heart endothelioid cells were used to determine the penetration rate of nitroblue tetrazolium (NitroBT) or other reactants into mitochondria in situ. Nitroblue formazan, produced as a consequence of succinate dehydrogenase activity in the mitochondria, was eluted and measured with a spectrophotometer. Prior injury of cells with hypo-osmolar solutions increased the rate of formazan production. Several methods are described or suggested for the statistical analysis of the data.

Animals↗
PubMed1982

Selection of optimum tetrazolium salts for use in histochemistry: the value of structure-staining correlations.

A structure-staining correlation study was made of a number of tetrazolium salts and formazans used in histochemistry. Numerical parameters describing molecular structure were calculated from structural formulae. Staining data were from previously published work. It was found that several significant practical variables (namely, uptake and reducibility of tetrazolium salts; substantivity, lipid solubility, and crystal size of formazans) could be predicted from structural parameters. Possible applications of these correlations are discussed: to increase our understanding of existing staining systems and to guide the design and selection of new tetrazolium salts for histochemical use.

Formazans↗
PubMedDate not supplied

Histochemical demonstration of creatine kinase activity using polyvinyl alcohol and auxiliary enzymes.

Creatine kinase activity (EC 2.7.3.2.) has been demonstrated in myocardium and skeletal muscle from rats by a method based on the incubation of cryostat sections with a polyvinyl alcohol-containing medium and the use of auxiliary enzymes. Hexokinase and glucose-6-phosphate dehydrogenase were spread on object glasses before mounting the sections to be incubated. In this way, the auxiliary enzymes were interposed between glass slide and section thus preventing loss of formazan generated within the sections. Creatine kinase activity was found to be localized in finely dispersed form along the myofibrils and as large granules in the sarcoplasm of myocardium and skeletal muscle. The formazan produced specifically by creatine kinase (test minus control), as measured cytophotometrically at 585 nm, was completely inhibited by 2 mM 2,4-dinitrofluorobenzene, a specific inhibitor of creatine kinase activity. The control reaction was unaffected by the inhibitor. The results obtained with the present method are similar to results obtained with the far more complicated semipermeable membrane technique. The introduction of auxiliary enzymes in the polyvinyl alcohol method enables the development of histochemical methods for many enzymes by linking the reactions to a dehydrogenase reaction.

Animals↗
PubMed1993

Determination of IL-2 and cytotoxicity of killer cells in MTT colorimetry.

The activity of interleukin 2 (IL-2) in culture supernatants of lymphokine-activated killer (LAK) cells and tumor infiltrating lymphocytes (TIL) as well as cytotoxicity of LAK cells on cultured leukemic cells were determined by MTT colorimetry. The results showed that higher activity of IL-2 in culture supernatant of LAK and TIL cells was found; it could be used to support the culture of IL-2 dependent cell lines. The significant cytotoxicity of LAK cells on leukemic cell lines could be found in vitro, and it was consistent with the ratio of effector cells to target cells. The number of living leukemic cells is consistently related with the concentration of formazan metabolite of MTT. It suggested that the numbers of living cells and cytotoxicity of LAK cells could be estimated by determination of formazan metabolite OD value.

Colorimetry↗