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Experiments have been carried out to explore the use of a tetrazolium salt, MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide in the detection of intracellularly generated superoxide in HeLa cells. From the use of a low molecular weight lipophilic mimic of superoxide dismutase, as well as superoxide dismutase, and inhibitors of superoxide dismutase, it is suggested that at least 20-30% of the intracellular reduction of MTT is due to superoxide. Whilst this may arise from mitochondria another possible intracellular source in HeLa cells may be xanthine oxidase. The overall rate of intracellular MTT reduction in HeLa cells is inversely dependent on levels of serum in the culture medium. Serum components with a modulatory role in this context are those with antioxidant function. Reduced MTT is also detectable extracellularly in cultures of HeLa cells and at least 80% of this is due to superoxide. Use of inhibitors suggest that whilst a small proportion (30%) may arise through an NADPH-oxidase type enzyme, other sources of extracellular superoxide in HeLa cells remain a possibility.
This article describes the use of a microdensitometer for the measurement of BPST formazan in tissue sections. BPST is a new tetrazolium salt, 2-(2-benzothiazolyl)-3-(4-phthalhydrazidyl)-5-styryl-tetrazolium chloride, which produces a single, well-defined formazan, and is thus easily quantified. The formazan gives an excellent localization, since BPST was originally designed for ultrastructural work. Activities are expressed in absolute units as n moles hydrogen/mm3, and are thus directly comparable with standard biochemical data.
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The Erlangen micro-lightguide spectrophotometer (EMPHO) was used to determine local mitochondrial activity. The measurement was based on a commercially available test (WST1, Boehringer Mannheim, Germany). A tetrazolium salt is cleaved to formazan by the 'succinate-tetrazolium reductase' system which belongs to the respiratory chain of the mitochondria and is active only in viable cells. 50 microliters of the reagent is placed in a recess at the tip of the lightguide. The tip of the lightguide is then placed at the tissue surface. The reagent diffuses into the cells and is cleaved to formazan. The absorption change is measured and analysed. This method was used in early chick embryos to determine the metabolic activity of the area vasculosa during the first 6 d of normoxic incubation. During this period of time the absorption change measured after 30 min increased from 0.001 units on day 2 to 0.262 units on day 6. This indicates an increase in local metabolic activity which may be due to an increase in metabolic activity of individual cells and/or an increase in the number of cells in the area under investigation. The results show that it is possible to determine local metabolic activity under in vivo conditions.
A simple method for estimation of the hydrophobic parameters (partition coefficient lg P and Hansch pi-value) of tetrazolium salts is reported using their relative velocities in reverse-phase layer chromatography. The experimental test values are close to relevant calculated values of the literature.
The enzyme cytochemical demonstration of glucose-6-phosphate dehydrogenase (G6PDH) with several tetrazolium salts has been studied with an artificial model of polyacrylamide films in corporated with the enzyme, which enabled teh correlation of cytochemical and biochemical data. In the model films no formazan could be produced with BPST and NT in contrast to NBT and TNBT. The reason of this phenomenon is discussed. The concentration of acrylamide strongly influenced the retention of the enzyme activity in the model films. The best results were obtained with a film matrix composed of 12% (w/v) acrylamide plus 0.6% (w/v) bisacrylamide. In this kind of film the retention of the enzyme activity was 59.0 +/- 5.4 (% +/- S.D.; n = 9). It was found that the biochemical and cytochemical (with TNBT) assay as applied to the model films both behave according to the Lambert-Beer law and that there was a high correlation between th biochemical and cytochemical data (r = 0.995).
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%.
A rapid colorimetric assay for viability of E. histolytica trophozoites using the tetrazolium salt MTT and phenazine methosulfate (PMS) was developed. The MTT is normally reduced by dehydrogenases of viable cells and transformed to formazan, a colored compound; the reaction is catalyzed by PMS. Trophozoites of E. histolytica were incubated in the presence of MTT and PMS during 15, 30, 45, 60 and 75 min, at 37 degrees C. Trophozoites killed by heat or cold were used as controls. The formazan produced into the cells was extracted with isopropanol-HCl and the optical density (OD) was measured at 570 nm. There was a linear relation between the number of viable trophozoites and the OD570 (r2 = 0.98). The trophozoites killed by cold or heat did not reduce MTT. The optimal time of incubation with MTT/PMS was 45 min. The MTT/PMS assay for determining viability of E. histolytica trophozoites is reproducible, rapid and easy to perform and it could be useful to study cytotoxicity and susceptibility to drugs.
Antibiograms of staphylococci were determined in a microtiter-system (fig. 1) within 4 h. The reactions could be clearly demonstrated by the use of tetrazolium salts. The procedure proved to be particularly useful for quantitative studies.
Allen, Emma G. (Downstate Medical School, State University of New York, Brooklyn). Use of tetrazolium salts for electron transport studies in meningopneumonitis. II. Reduced nicotinamide adenine dinucleotide phosphate system. J. Bacteriol. 92:1041-1046. 1966.-The conditions of electron transfer from nicotinamide adenine dinucleotide phosphate in meningopneumonitis (MP) are described and compared with electron transfer from nicotinamide adenine dinucleotide by this organism. The observations suggest that, in either system, there may be more than one pathway of electron flow, and that these pathways differ from those in normal membrane particulates. It was also found that after trypsin treatment, particulates from pools of normal allantoic fluids and membranes retain the normal characteristics, whereas those from pools of MP-infected fluids and membranes assume the characteristics of MP particles from fluids.
Current methodology to determine absence of live mycobacteria in tuberculin purified protein derivative (PPD) takes up to 8 weeks to perform and may also involve testing on animals. In this paper we describe an in vitro test utilising the tetrazolium salt, 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide (XTT) to monitor the absence of live Mycobacterium tuberculosis (Mtb) in PPD. In the presence of live cells XTT is converted to a coloured formazan product that can be measured spectrophotometrically. Live mycobacteria present in spiked PPD were detected by a marked change in optical density above background levels. This test is easy to perform and is complete in just 48 hr.
The Escherichia coli membrane-bound D-lactate dehydrogenase and succinate dehydrogenase were assayed on the basis of the phenazine methosulfate- (PMS-) mediated reduction of the tetrazolium salt, MTT. An initial slower phase (lag) in the time-course of the reaction was observed and analyzed. The results were as follows. (1) The time lag in the assay of the D-lactate dehydrogenase was eliminated by preincubating the membranes with PMS plus D-lactate, with PMS plus succinate, or with PMS plus NADH (conditions which implicated PMS reduction). (2) When the D-lactate dehydrogenase was assayed by another method based on the measurement of the pyruvate formed, neither was a time lag observed nor was the enzyme activity affected by membrane preincubation with PMS plus D-lactate. (3) Although the superoxide radical was involved in MTT reduction, this radical seemed not to participate in the generation of the time lag. (4) Membranes whose D-lactate dehydrogenase activity had previously been destroyed by heating at 80 degrees C for 1 min, were able to prolong the time lag in MTT reduction when added to the assay medium for the D-lactate dehydrogenase from untreated membranes, whereas membranes previously heated at 100 degrees C instead of 80 degrees C did not have this effect. It was concluded that the E. coli membranes interfered in the dehydrogenase assay based on the PMS-mediated reduction of MTT. The time lag was interpreted as a period during which the interfering substance reacted with reduced PMS inhibiting the reduction of MTT.
A tissue culture bioassay, using the mouse neuroblastoma cell line (Neuro2A), was improved to provide a simple and sensitive bioassay for TTX or sodium channel-blocking toxins (SCB). The water-soluble tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetraz olium, monosodium salt (WST-1), was applied to replace the time-consuming and subjective cell-counting procedure of the cells with automatic measurement, using a microplate reader. It was also confirmed that this method is directly applicable to bacterial culture supernatants, with the precaution of possible interference.
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Mouse bone marrow cells were grown in liquid culture in microtiter plates in the presence of different colony-stimulating factors (CSF). Growth was assayed using the tetrazolium salt MTT, which is reduced in the mitochondria of viable cells to a water-insoluble blue formazan dye. Two technical problems have limited the use of this assay: the solubilization of the dye crystals and the necessity to acidify the phenol red in the culture medium. Both could be solved here by the use of a developing solution of 5% formic acid in isopropanol. Using manual mixing combined with a short sonication by floating the plates in a sonic bath, the crystals were dissolved within minutes. There was no flocculation of protein, even using medium with 20% serum. The color remained stable for at least 4 h. This enabled the semi-automatic measurement of large numbers of cultures directly in the microtiter plates. Growth and differentiation of myelopoietic precursor cells in the liquid cultures was shown to be comparable to that in soft agar. Cell growth was CSF-dependent. The calculated cell yield per colony forming cell (CFC) seeded was within the range of the average cell number per colony found in soft agar, and the spectrum of mature cells obtained reflected the type of CSF used as stimulus. Using the combined culture and assay systems, it was possible to perform detailed kinetic studies of myelopoiesis. This technique should be useful for studying the mechanisms of action of pharmacological modulators of myelopoiesis.
Since the first report in 1983, blood platelet reactivity from patients with immediate hypersensitivity reactions, and particularly allergic asthma and aspirin-sensitive asthma, has been evaluated by the release of cytotoxic mediators able to induce the death of parasite larvae, and by the generation of oxygen derived free radicals, measurable by chemiluminescence. We demonstrate here that platelets are able to reduce MTT tetrazolium salt proportionally to the stimulation level in two models of platelet triggering, one IgE-dependent and one aspirin-dependent. This method correlated significantly with the cytotoxicity assay of platelet stimulation (r = 0.965, p < 10(-5) for IgE-dependent stimulation; r = 0.723, p < 10(-4) for aspirin-dependent stimulation). The MTT colorimetric assay should complement or possibly replace previous methods of assessing platelet activation which were of limited use allowing broader investigations on the involvement of platelets in immune processes and in allergic or pseudoallergic reactions.
One of the indicators of non-specific, cell-mediated immunity can be the ability of phagocytic system to react to the presence of antigenic impulses and stimulators. The phagocytic activity induced in this way in vitro can be evaluated quantitatively by help of different methods. In this study the method of the reduction of colourless tetrazolium salt (INT) to the red formazane was used. The optimum conditions for carrying out this test in isolated peripheral pig leucocytes were determined. It was stated that 3-4 ml samples of pig blood, from which 6.10(6) leucocytes necessary for the test can be isolated, were sufficient for the examination of phagocytic activity. Further, 45 minutes were determined as the optimum time of incubation It was found that the INT test can be reproduced by 24 hours after blood sampling provided the blood is kept at the temperature of +4 degrees C. The INT test was used for examining 38 pig blood samples before and after myostress; this stress has a significant effect both on a decrease of phagocytic activity and on the ability of leucocytes to migrate in the LMI test. The INT test was further used for examining pigs before the administration of the first dose of the inactivated vaccine against the Aujeszky's disease virus and two days after; the application of the vaccine significantly increased the phagocytic activity.