Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Formazans”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 361 records · Page 20Linked to original sources

Field trials of a rapid test for G6PD deficiency in combination with a rapid diagnosis of malaria.

A rapid single-step screening method for detection of glucose-6-phosphate dehydrogenase (G6 PD) deficiency was evaluated on Halmahera Island, Maluku Province, Indonesia, and in Shan and Mon States, Myanmar, in combination with a rapid diagnosis of malaria by an acridine orange staining method. Severe deficiency was detected by the rapid test in 45 of 1126 volunteers in Indonesia and 54 of 1079 in Myanmar, but it was difficult to distinguish blood samples with mild deficiency from those with normal activity. 89 of 99 severely deficient cases were later confirmed by formazan ring method in the laboratory, but 5 with mild and 5 with no deficiency were misdiagnosed as severe. Of the samples diagnosed as mild and no deficiency on-site, none was found to be severely deficient by the formazan method. Malaria patients were simultaenously++ detected on-site in 273 samples on Halmahera island and 277 samples from Shan and Mon States. In Mon State, primaquine was prescribed safely to G6 PD-normal malaria patients infected with Plasmodium vivax and/or gametocytes of P. falciparum. The new rapid test for G6 PD deficiency may be useful for detecting severe cases under field conditions, and both rapid tests combined are can be useful in malaria-endemic areas, facilitating early diagnosis, prompt and radical treatment of malaria and suppression of malaria transmission.

Acridine Orange↗

Beta-amyloid-induced cell toxicity: enhancement of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-dependent cell death.

In an attempt to understand the cause of neurodegeneration in Alzheimer's disease, the toxic effects of beta-amyloid (Abeta) peptides have been widely studied. At high micromolar concentrations Abeta peptides have been demonstrated to be acutely toxic to various cell types. At submicromolar concentrations, Abeta peptides have been suggested to inhibit cellular metabolic activity, due to their inhibition of the ability of cells to metabolize the oxidoreductase substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Here we show, first, that MTT reduction surprisingly leads to a breakdown in PC12 cell membrane integrity and cell death, presumably through the formation of a crystalline formazan product, and, second, that pretreatment of PC12 cells with nanomolar concentrations of Abeta peptide, rather than inhibiting their metabolic activity, increases the susceptibility of these cells to the secondary toxic effect of formazan crystal formation. These results suggest that low nanomolar concentrations of Abeta render membranes more susceptible to damage by a secondary insult, in this case, MTT reduction. It is plausible that such an effect, when combined with additional risk factors, could contribute to the neurodegeneration that occurs in Alzheimer's disease.

Amyloid beta-Peptides↗

Mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction is one of the most frequently used methods for measuring cell proliferation and neural cytotoxicity. It is widely assumed that MTT is reduced by active mitochondria in living cells. By using isolated mitochondria from rat brain and B12 cells, we indeed found that malate, glutamate, and succinate support MTT reduction by isolated mitochondria. However, the data presented in this study do not support the exclusive role of mitochondria in MTT reduction by intact cells. Using a variety of approaches, we found that MTT reduction by B12 cells is confined to intracellular vesicles that later give rise to the needle-like MTT formazan at the cell surface. Some of these vesicles were identified as endosomes or lysosomes. In addition, MTT was found to be membrane impermeable. These and other results suggest that MTT is taken up by cells through endocytosis and that reduced MTT formazan accumulates in the endosomal/lysosomal compartment and is then transported to the cell surface through exocytosis.

Animals↗

Ischemic preconditioning of rat livers against cold storage-reperfusion injury: role of nonparenchymal cells and the phenomenon of heterologous preconditioning.

Brief periods of ischemia followed by reperfusion render tissues resistant against subsequent prolonged ischemia, a phenomenon called ischemic preconditioning. The effect of ischemic preconditioning on liver transplantation was investigated in relation to sinusoidal endothelial cell injury and Kupffer-cell activation, which are prominent features of storage and reperfusion injury leading to liver graft failure. Rat livers were preconditioned by 5 or 10 minutes of ischemia and 5 minutes of reperfusion and stored in University of Wisconsin (UW) solution for 30 hours. Livers were then reperfused for 15 minutes with physiological buffer containing trypan blue. Under these conditions, injury occurs predominantly to sinusoidal endothelial cells, reflected by trypan blue staining of nonparenchymal cells in histological sections. Ischemic preconditioning decreased nonparenchymal cell killing by more than 50%. When half the liver was preconditioned, sinusoidal endothelial cells were also protected in the contralateral half. Other stored livers were reperfused with nitroblue tetrazolium, which is converted to insoluble formazan by superoxide radicals. Ischemic preconditioning decreased the intensity of formazan deposition over Kupffer cells. Finally, stored livers were transplanted into nontreated rats. Ischemic preconditioning improved recipient long-term survival after 30 hours of cold ischemic storage in UW solution from 30% to 80% and decreased serum tumor necrosis factor-alpha levels in posthepatic blood 4 hours postoperatively from 98 to 54 pg/mL. In conclusion, ischemic preconditioning protects sinusoidal endothelial cells and suppresses Kupffer-cell activation after storage and reperfusion. As a result, graft survival improves after liver transplantation. Moreover, ischemia to half the liver confers protection to the other half. Such heterologous preconditioning provides a new means to protect liver tissue against ischemia-reperfusion injury without imposing ischemia on the target tissue.

Animals↗

Interference of plant extracts, phytoestrogens and antioxidants with the MTT tetrazolium assay.

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a widely used screening method to measure cell viability and proliferation. When testing the effects of kaempferol on breast cancer cell number (crystal violet staining) and viability (MTT tetrazolium assay) conflicting results were obtained. Cell number decreased but MTT formazan formation increased, suggesting a direct interaction of kaempferol with the MTT tetrazolium reduction. Direct reductive potential was observed in a cell-free system for the presumptive phytoestrogens kaempferol and resveratrol, and extracts of Hypericum perforatum L. and Cimicifuga racemosa L. All agents led to instantaneous dark blue formazan formation in the absence of cells. Additionally, antioxidants such as ascorbic acid, vitamin E and N-acetylcysteine interfered with the MTT tetrazolium assay. When MCF7 and HS578 cells treated with kaempferol were washed before addition of MTT tetrazolium, the direct reduction of dye was reduced significantly. These results indicate that the MTT tetrazolium assay may lead to false positive results when testing natural compounds with intrinsic reductive potential.

Acetylcysteine↗

A quantitative nitroblue tetrazolium assay for determining intracellular superoxide anion production in phagocytic cells.

Conventionally, a semi-quantitative microscopic nitroblue tetrazolium (NBT) assay is used to determine the production of superoxide anion (O2(-)) in various phagocytic cells. This microscopic assay is conducted by counting the cells containing blue NBT formazan deposits, which are formed by reduction of the membrane permeable, water-soluble, yellow-colored, nitroblue tetrazolium (Y-NBT) by O2(-). However, this assay is semi-quantitative and is prone to observer bias. In the present study, we modified the NBT assay by dissolving the blue formazan particles using 2M potassium hydroxide and dimethylsulfoxide and then measured its absorbance using a microplate reader at 620nm. The absorbance of dissolved NBT increased in proportion to cell number (r = 0.9907), incubation time, and stimulus concentration. To test the usefulness of this modified assay, we compared the abilities of a number of types of phagocytic cells to produce O2(-). The cells examined included murine macrophage cell lines (RAW 264.7 and J774), freshly prepared murine peritoneal macrophages and neutrophils, a human myeloid cell line (PLB-985), and freshly prepared human peripheral blood neutrophils. In addition, we demonstrate that nitric oxide produced by RAW 264.7 cells does not interfere with the modified colorimetric NBT assay. Taken together, our results indicate that the modified colorimetric NBT assay is simple, sensitive, and quantitative, and that it can be used to determine the amounts of intracellular O2(-) produced by phagocytic cells. Thus, this assay is sensitive enough to measure, quantitatively, even the small amounts of O2(-) produced in monocytes and macrophages that are not detectable by the conventional microscopic NBT assay.

Animals↗

The cytochemical localization of oxidative enzymes. II. Pyridine nucleotide-linked dehydrogenases.

Methods are presented for the intramitochondrial localization of various diphosphopyridine nucleotide and triphosphopyridine nucleotide-linked dehydrogenases in tissue sections. The cytochemical reactions studied involve the oxidation of the substrates by a specific pyridino-protein. The electron transfer of tetrazolium salt is mediated by the diaphorase system associated with the dehydrogenase. The final electron acceptor was either p-nitrophenyl substituted ditetrazole (nitro-BT) or N-thiazol-2-yl monotetrazole (MTT), the latter giving rise to metal formazan in the presence of cobaltous ions. Mitochondrial localization of the formazan precipitate could be achieved by using hypertonic incubating media containing high concentrations of substrate and co-enzyme. A fast reduction of tetrazolium salt was obtained by chemically blocking the respiratory chain enzymes beyond the flavoproteins. Although diaphorase systems are implicated in the reduction of tetrazolium salts, specific dehydrogenases are solely responsible for the distinct distribution pattern obtained in tissues with various substrates. The present findings in tissue sections are discussed in conjunction with existing biochemical evidence from differential centrifugation experiments.

Dihydrolipoamide Dehydrogenase↗

A cytochemical study of the dehydrogenases of mitochondria and mitochondrial particulates by a monotetrazolium-cobalt chelation method.

In one of the current histochemical methods for dehydrogenases and diaphorases the final product is a metal-formazan dye derived from reduction of an N-thiazolyl-substituted tetrazolium. Sites of enzymic activity consistently appear as intramitochondrial dots 0.2 to 0.3 micro in diameter. When applied to active particles from disrupted mitochondria (Keilin-Hartree preparation, electron transport particle, Cooper-Lehninger particle) the individual particles appear as black dots 0.1 to 0.3 micro in diameter. It is clear that formazan is deposited progressively upon the particles and the results suggest that the latter may be spatially arranged in mitochondria so that areas of activity are separated by quiescent regions.

Cobalt↗

Lycopene I--effect on osteoclasts: lycopene inhibits basal and parathyroid hormone-stimulated osteoclast formation and mineral resorption mediated by reactive oxygen species in rat bone marrow cultures.

Osteoclasts have been shown to produce reactive oxygen species (ROS) that can stimulate bone resorption. We explored the hypothesis that lycopene, the antioxidant carotenoid from tomatoes, can inhibit mineral resorption by inhibiting osteoclast formation and the production of ROS. Cells from bone marrow prepared from rat femur were plated into 16-well calcium phosphate-coated Osteologic Multi-test Slides and cultured in alpha-minimal essential medium supplemented with dexamethasone, beta-glycerophosphate, and ascorbic acid. The cells were treated with varying doses of lycopene in the absence or presence of parathyroid hormone (PTH) at the start of culture and at each medium change (i.e., every 48 hours). On day 8, mineral resorption pits were quantitated. Similar, parallel experiments were carried out in 12-well plastic dishes to assess tartrate-resistant acid phosphatase (TRAP) activity. Results showed that lycopene inhibited TRAP + formation of multinucleated cells in both vehicle- and PTH-treated cultures. Osteoclasts reduced nitroblue tetrazolium (NBT) to purple-colored formazan, indicating the presence of ROS in these cells. The formazan-staining cells were decreased by treatment with 10(-5) M lycopene, indicating that lycopene inhibited the formation of ROS-secreting osteoclasts. In conclusion, we have shown that lycopene inhibits basal and PTH-stimulated osteoclastic mineral resorption and formation of TRAP + multinucleated osteoclasts, as well as the ROS produced by osteoclasts. These findings are novel and may be important in the pathogenesis, treatment, and prevention of osteoporosis.

Acid Phosphatase↗

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.

Cell Line↗

A tetrazolium-based colorimetric assay for titration of neutralizing antibodies against vaccinia virus.

A colorimetric assay for titration of neutralizing antibodies against vaccinia virus was developed. The test is based on the ability of live cells in culture to reduce the yellow tetrazolium salt MTT (thiazolyl-blue), to its blue formazan derivative. Antisera from individuals vaccinated with vaccinia virus against smallpox were serially diluted, incubated with 100 plaque-forming units (PFU) of vaccinia virus for 1 hour at 37 degrees C, and then transferred to a 96-well plate containing monolayers of B-SC-1 cells. After incubation for 3 to 4 days at 37 degrees C, when more than 80% of the control infected cultures exhibited high degree of cytopathogenic effect, MTT was added. The absorbance of the formazan formed and extracted by dimethylsulfoxide was read at 492 nm by an automatic microplate spectrophotometer. A good correlation was found between the results obtained using this newly developed method and those of the plaque-reduction assay.

Antibodies, Viral↗

Colorimetric measurement of plasma lactate.

A rapid and simple enzymatic method for plasma lactate is proposed using stable reagents to produce the formazan color. This method agrees well with a reference kit method, r = 0.955 and the regression equation is y = 0.99x + 0.09. The over-all recovery averages 100%, with a precision ranging from 0.6 to 3.3%. No interferences have been shown with the formazan reaction. The proposed method is inexpensive, ideal for batch analyses, and is an attractive method for the busy clinical laboratory.

Adenosine Monophosphate↗

Phorbol diacetate inhibits superoxide anion radical production and tumor promotion by mezerein.

The ability of the non-promoter phorbol diacetate (PDA) to modulate superoxide anion radical production by the complete tumor promoter phorbol myristate acetate (PMA) or the second stage promoter mezerein was assessed. Superoxide anion radical production was measured by the superoxide dismutase inhibitable reduction of nitroblue tetrazolium (NBT) to a blue intracellular formazan precipitate. These studies demonstrated that superoxide anion radical production by murine peritoneal exudate cells (PEC) stimulated by i.p. injection with mezerein (100 ng) is inhibited in a dose-dependent manner by co-administration with PDA (1-1000 ng). There was no effect on the number of formazan-positive PEC when PDA was co-administered with PMA. In a two-stage tumor promotion bioassay in female SENCAR mice initiated with 25.6 micrograms dimethylbenz[a]anthracene (DMBA) followed by first stage promotion with PMA (4X, 2 micrograms), co-administration of mezerein (2 micrograms) with 2 micrograms or 20 micrograms PDA reduced the number of papillomas after 14 weeks by 38% and 44%, respectively, compared with mezerein treatment alone. PDA (20 micrograms) when co-administered with mezerein (2 micrograms) does not inhibit mezerein induced hyperplasia in mouse skin. These results suggest a correlation between the ability of PDA to inhibit both superoxide anion radical production and tumor promotion by mezerein.

Animals↗

A rapid colorimetric assay of fungal viability with the tetrazolium salt MTT.

A rapid colorimetric assay for viability of fungi that uses the tetrazolium salt MTT is described. All live species of a wide range of medically important fungi that were tested reduced MTT, with resultant intracellular formation within several hours of clearly visible purple crystals of MTT-formazan. The assay proved particularly useful in measuring viability of individual hyphae; greater than 99% of live Aspergillus fumigatus and Rhizopus oryzae hyphae stained purple when incubated with MTT compared with 0% of hyphae killed by any of a variety of methods. The MTT method for measuring fungal viability closely correlated with a slide-culture technique when killing of A. fumigatus hyphae by graded concentrations of hydrogen peroxide was compared. MTT-formazan was easily extracted from the fungi by alcohol, a procedure allowing spectrophotometric quantification of fungal killing in both cell-free and cellular systems. A linear relation between inocula and MTT reduction of several fungi, including Candida albicans blastospores and A. fumigatus hyphae, was demonstrated. Human leukocytes killed 80.3% of C. albicans blastospores as measured by the MTT assay, compared with 70.5% as measured by dilutions and pour plates. Killing of A. fumigatus hyphae by human leukocytes was also quantitated by using the MTT assay.

Fungi↗

Generation of superoxide anion and localization of CuZn-superoxide dismutase in the vascular tissue of spinach hypocotyls: their association with lignification.

The sites of generations of superoxide anions and hydrogen peroxide in cross sections of hypocotyls from spinach seedlings were located by staining with nitroblue tetrazolium (NBT) and with starch-iodide, respectively. Formazan, produced upon the reduction of NBT by superoxide, was observed mainly in the vascular tissue only in the presence of inhibitors of CuZn-superoxide dismutase (CuZn-SOD), and its formation was suppressed under anaerobic conditions. Thus, NBT was reduced to formazan specifically by the superoxide anions generated in vascular tissue. The reduction of NBT was suppressed by inhibitors of NAD(P)H oxidase, but neither by cyanide nor azide, indicating the involvement of NAD(P)H oxidase in the generation of superoxide anions in the vascular tissue. Starch-I2 complex also was formed in the vascular tissue, but not in the presence of either the CuZn-SOD inhibitor or the NAD(P)H oxidase inhibitor, indicating that the hydrogen peroxide is produced via the catalytic disproportionation with CuZn-SOD of the superoxide generated by NAD(P)H oxidase. Generations of superoxide anions and hydrogen peroxide in the vascular tissue were particularly apparent in the xylem and associated with the sites of distribution of CuZn-SOD as determined by an immunohistochemical method, and also with the location of lignin as determined by the phloroglucin-HCl reaction.

Enzyme Inhibitors↗

Ultrastructural distribution of NADPH-diaphorase in cortical synapses.

To elucidate the role of nitric oxide (NO) in synaptic structures, the ultrastructural localization of the enzyme NO synthase (NOS), based on the cytochemical NADPH-diaphorase staining, was studied in neocortical and hippocampal neuropil areas. For the localization of NADPH-diaphorase activity a special tetrazolium salt (BSPT) was applied. BSPT-formazan, the osmiophilic reaction product, was found to be attached to endomembranes, predominantly the endoplasmic reticulum. Quantitative studies on synaptic regions revealed that mainly presynaptic areas (41% in hippocampus, 38% in neocortex) showed labelling. Postsynaptic regions were only exceptionally labelled by BSPT-formazan (3% in hippocampus, 5% in neocortex). The present findings support the view that NO may be involved in diverse synaptic functions acting preferentially from the presynaptic side.

Animals↗

Generation of intracellular active oxygens in mouse FM3A cells by 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole, the activated Trp-P-2.

Mouse FM3A cells in culture were treated with a reactive metabolite of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2-(NHOH]. When the treated cells, which were judged as viable on the basis of trypan-blue exclusion, were subjected to nitroblue tetrazolium staining, formazan was formed inside the cells, a fact suggesting the intracellular presence of superoxide. No formazan formation was detected on treatment of the cells with Trp-P-2. Single-strand breaks in the cellular DNA took place during this treatment with Trp-P-2(NHOH). Since Trp-P-2(NHOH) in solution generates superoxide anion accompanying its oxidative degradation, we conclude that the Trp-P-2(NHOH) treatment produces intracellular active oxygens that can damage DNA.

Animals↗

A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual erythrocytes. II. Further improvements of the staining procedure and some observations with glucose-6-phosphate dehydrogenase deficiency.

A cytochemical method for staining glucose-6-phosphate dehydrogenase (G6PD) activity in individual erythrocytes as reported previously has been optimized further by the incorporation of a number of technical improvements. Analysis of the enzyme content in erythrocytes of normal individuals as well as patients suffering from G6PD deficiency in the homozygous and heterozygous forms allows these three categories to be easily distinguished. Considerable formazan production occurs in most erythrocytes of a healthy person and only a small percentage of the cells appeared to be negative. Two cell populations of almost equal size could be discerned in heterozygotes for G6PD deficiency, one completely negative, the other with a variable amount of formazan per cell. Homozygous deficiency leads to a population of negative cells with a few positive ones after staining. It is concluded that a reliable method has been found for analysis of G6PD deficiency in erythrocytes at the single cell level.

Erythrocytes↗