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PubMed1988

Early morphological changes in the striated muscles in normal and dystrophic chickens.

Histological and histochemical analyses were performed on the anterior latissimus dorsi muscle (ALD, red muscle) and the posterior latissimus dorsi muscle (PLD, white muscle) in normal (line 412) and dystrophic chickens (line 413) from 19 day embryos to 6 weeks of age. PLD, the white muscle, in dystrophic chickens showed higher percentages of red and intermediate fibres than those of normal chickens during the early development of muscles. Increases of the oxidative enzyme activities and the numbers of NADH--TR formazan granules in the white fibres of PLD were already found at 1 week of age in dystrophic chicken. Fibre types, oxidative enzyme activities and NADH--TR formazan granules showed no differences in ALD between normal and dystrophic chickens. These results suggest that increases of oxidative enzyme activities and formazan granule numbers and incomplete fibre type differentiation in PLD of dystrophic chickens are early pathological processes in such birds.

Animals↗
PubMedDate not supplied

Comparison of different techniques for the postmortem diagnosis of myocardial infarction.

A study was performed on 74 medicolegal autopsy cases for the purpose of comparing the reliability of four different techniques (haematoxylin-eosin stain, acridine orange method, formazan test and K/Na ratio) used for the postmortem diagnosis of myocardial infarction. Although the results showed that the formazan test and K/Na ratio give a good reliability to rule out a myocardial infarction, the most specific technique for the diagnosis of infarct is the formazan test.

Acridine Orange↗
PubMed1999

Both oxidative stress-dependent and independent effects of amyloid beta protein are detected by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction assay.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay has been widely used for evaluating amyloid beta protein (Abeta) toxicity. However, the potency of Abeta in inhibiting cellular MTT reduction and the underlying mechanism have been reported with some discrepancies among researchers. To understand what makes such discrepancies, the effect of Abeta detected by MTT reduction assay was re-examined in detail by using cultured rat hippocampal neurons. Micromolar concentrations (>10 microM) of Abeta caused a decrease in cell viability, which resulted in a decrease in MTT reduction per well regardless of assay time. The micromolar Abeta-induced decrease of cellular MTT reduction was significantly attenuated by antioxidants (catalase, propyl gallate or Trolox). On the other hand, nanomolar Abeta did not affect cellular MTT reduction activity at an initial stage of assay (<1 h), and decreased the total production of MTT formazan by accelerating the exocytosis of MTT formazan when MTT assay was performed for a longer time (>2 h). The assay time-dependent, nanomolar Abeta-induced decrease of cellular MTT reduction was not at all affected by antioxidants. Furthermore, subtoxic concentration of H2O2 failed to mimic the effect of nanomolar Abeta on MTT reduction. These results indicate that micromolar Abeta-induced, oxidative cell death is detected by MTT assay regardless of assay time, whereas nanomolar Abeta-induced acceleration of MTT formazan exocytosis is not mediated by oxidative stress and detected only when MTT assay is performed for a longer time. The time of MTT assay should be properly chosen depending on the purpose of the study.

Amyloid beta-Peptides↗
PubMedDate not supplied

The value of succinate dehydrogenase stain in the post-mortem diagnosis of early acute myocardial infarction. A forensic study.

In consecutive medicolegal material comprising 79 persons, the formazan test revealed recent myocardial infarction in 16 cases (20%). Only 4 of these cases (25%) were demonstrated by macroscopic examination. By means of microscopic examination, the 4 infarctions recognized macroscopically and 5 other cases which were demonstrated by the formazan test could be confirmed. If the formazan test is employed as an independent demonstration of myocardial infarction, a total of 16 cases could be demonstrated (as mentioned above), or four times as many as at macroscopic examination at autopsy.

Acute Disease↗
PubMed1998

Amyloid beta peptide alters intracellular vesicle trafficking and cholesterol homeostasis.

Amyloid beta peptide (Abeta) is thought to play a central role in the pathogenesis of Alzheimer disease (AD). How Abeta induces neurodegeneration in AD is not known. A connection between AD and cholesterol metabolism is suggested by the finding that people with the apolipoprotein E4 allele, a locus coding for a cholesterol-transporting lipoprotein, have a modified risk for both late-onset AD and cardiovascular disease. In the present study we show that both Abeta and submicromolar concentrations of free cholesterol alter the trafficking of a population of intracellular vesicles that are involved in the transport of the reduced form of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT formazan), the formation of which is a widely used cell viability assay. Treatments that change cellular free cholesterol levels also modulate the trafficking of the MTT formazan-containing vesicles, suggesting that the trafficking of these vesicles may be regulated by free cholesterol under physiological conditions. In addition, Abeta decreases cholesterol esterification and changes the distribution of free cholesterol in neurons. These results suggest that the MTT formazan-transporting vesicles may be involved in cellular cholesterol homeostasis and that the alteration of vesicle transport by Abeta may be relevant to the chronic neurodegeneration observed in AD.

Amyloid beta-Peptides↗
PubMed2006

Evaluation of MTT assay for measurement of emodin-induced cytotoxicity.

The specificity and sensitivity of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium assay can be influenced by certain factors, such as colored substances and cell volume. When the MTT assay is applied to measure cytotoxicity induced by emodin, its accuracy might be affected by emodin itself. Vascular smooth muscle cells were cultured in M199 medium. The optical density of emodin or formazan was measured by spectrophotometry. Emodin has a different absorption spectrum in different solvents. The solvents containing water induced a red shift of the absorption curve of emodin, which increased the overlap of the absorption curves of emodin and formazan. Formazan was formed from the MTT tetrazolium salt by emodin in a dose-dependent manner, which was partially suppressed by serum. Cytotoxicity was induced by emodin in a time- and dose-dependent manner in a modified MTT assay. The data suggest that emodin can alter the accuracy of the MTT assay but that a modified MTT assay is still valuable in measuring emodin-induced cytotoxicity.

Cell Line↗
PubMed1993

Factors affecting the selection and use of tetrazolium salts as cytochemical indicators of microbial viability and activity.

The reduction of four tetrazolium cations (TCs), nitroblue tetrazolium (NBT), neotetrazolium (NT), methylthiazolyldiphenyltetrazolium (MTT) and iodonitrophenyltetrazolium (INT), by viable micro-organisms, immobilized on glass cover slips, was examined by light microscopy with a view to determining a systematic basis for applying these reagents as cytochemical indicators of microbial viability and activity. The potential value of histochemical information about TC reactions for developing their microbiological applications was also assessed. INT and MTT detected viable cells more readily than NBT and NT. In order to obtain cell-localized formazan, MTT required cobalt ions in the reaction mixture and INT reactions had to be assessed soon after mounting. In general, formazan deposition could be accelerated by the addition of glucose and an intermediate electron carrier (IEC) to the reaction mixture, although inhibitory effects of IECs were also detected. Cultures in exponential phase, in stationary phase and inhibited by chloramphenicol could be differentiated with MTT but not with INT. For some organisms, notably Candida albicans, Pseudomonas aeruginosa and Enterococcus faecalis. TC reactions proved to be a relatively insensitive means of demonstrating viability. Two parameters used in selecting TCs for histochemical reactions, lipophilicity and reducibility appeared to be predictive for the relative sensitivity of these reagents as indicators of cell viability. The concepts of substantivity, a measure of non-specific interactions between reagents and staining substrates, and TC oxygen sensitivity, the effect of competition between oxygen and TCs for electrons, were found to be relevant to formazan deposition in live microbes. These findings support the use of TCs as cytochemical probes of microbial activity in defined settings and the use of histochemical knowledge to support further development of these techniques.

Bacteria↗
PubMed1996

Effects of substrates and phosphate on INT (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride) and CTC (5-cyano-2,3-ditolyl tetrazolium chloride) reduction in Escherichia coli.

The effects of substrates of primary aerobic dehydrogenases, and inorganic phosphate on aerobic INT and CTC reduction in Escherichia coli were examined. In general, INT produced less formazan than CTC, but INT (+) cell counts remained near values of CTC (+) cells. INT and CTC (+) cell numbers were higher than plate counts on R2A medium using succinate, formate, lactate, casamino acids, glucose, glycerol (INT only) and no substrate. Formate resulted in the greatest amount of INT and CTC formazan. Reduction of both INT and CTC was inhibited above 10 mmol l-1 phosphate, and this appeared to be related to decreased rates of O2 consumption. Formation of fluorescent CTC (+), but not INT (+) cells was also inhibited in a concentration dependent manner by phosphate above 10 mmol l-1. From light microscopic observations it appeared CTC formed increasing amounts of poorly or non-fluorescent formazan with increasing phosphate. Therefore, use of phosphate buffer in excess of 10 mmol l-1 may not be appropriate in CTC and INT reduction assays.

Colony Count, Microbial↗
PubMed2001

The sites of interaction of triphenyltetrazolium chloride with mitochondrial respiratory chains.

The inability of cells and microorganisms to reduce the colourless electron acceptor triphenyltetrazolium chloride (TTC) to a red formazan precipitate is commonly used as a means of screening for cells that have a dysfunctional respiratory chain. The site of reduction of TTC is often stated to be at the level of cytochrome c oxidase where it is assumed to compete with oxygen for reducing equivalents. However, we show here that TTC is reduced not by cytochrome c oxidase but instead by dehydrogenases, particularly complex I, probably by accepting electrons directly from low potential cofactors. The reduction rate is fastest in coupled membranes because of accumulation in the matrix of the positively charged TTC+ cation. However, the initial product of TTC reduction is rapidly reoxidised by molecular oxygen, so that generation of the stable red formazan product from this intermediate occurs only under strictly anaerobic conditions. Colonies of mutants defective in cytochrome oxidase do not generate sufficiently anaerobic conditions to allow the intermediate to form the stable red formazan. This revision of the mode of interaction of TTC with respiratory chains has implications for the types of respiratory-defective mutants that might be detected by TTC screening.

Anaerobiosis↗
PubMed2003

3-nitropropionic acid inhibition of succinate dehydrogenase (complex II) activity in cultured Chinese hamster ovary cells: antagonism by L-carnitine.

3-Nitropropionic acid (3-NPA) is an inhibitor of the mitochondrial enzyme succinate dehydrogenase (SDH, a part of complex II) that links the tricarboxylic acid (TCA) cycle to the respiratory electron transport chain. 3-NPA inactivates SDH by covalently and irreversibly binding to its active site. We previously examined the effects of 3-NPA on the histochemical activity of SDH in vivo, by using the reduction of a yellow tetrazolium dye (nitro blue tetrazolium) to a blue formazan as an indicator. In studies of cultured cells, the related dye methylthiazoletetrazolium (MTT) has commonly been used as an indicator of the presence and number of viable cells; that is cells that are capable of producing energy via the TCA cycle. Here we observed that doses of 3-NPA as low as 10(-8) M inhibited formazan production in an in vitro model system using CHO cells. This effect was antagonized by l-carnitine, which greatly increased the production of formazan, indicating a considerable improvement in energy production by the cultured cells. CHO cells appear to be a convenient model for the evaluation of therapeutic compounds that may modulate cellular bioenergetics.

Animals↗
PubMed1976

Quantitative reduction of 2,3,4-triphenyl tetrazolium chloride by hamster trachea organ cultures: effects of Mycoplasma pneumoniae cells and membranes.

The ability of Mycoplasma pneumoniae cells and membranes to affect tetrazolium reduction by hamster trachea organ cultures was evaluated. Uninfected trachea explants reduced 2,3,5-triphenyl tetrazolium chloride (TTC) and nitro-blue tetrazolium when incubated at 37 C in the absence of air. Reduced tetrazolium salts (formazans) were extractable with acetone or ethylene glycol and could be quantitated spectrophotometrically. The optimal assay system involved the use of three or more tracheal rings incubated for 2 h in 0.12% TTC in Tyrode balanced salts supplemented with 1.2% sodium succinate. Formazan was extracted for 5 min with acetone, and the optical density (490 nm) was determined. Trachea explants with metabolic activity reduced or obliterated by freeze-thaw lysis, heat (56 C X 30 min), or cyanide (0.1 M NaCN X 30 min) had negligible ciliary activity and tetrazolium reduction activity (optical density at 490 nm [dry weight]). Tracheas exposed to mycoplasma cells or membranes also showed significantly decreased ciliary activity and tetrazolium reduction; e.g., only 5pc of the ciliary activity and reduction capacity remained after 5 days in culture when infected with M. pneumonia PI 1428 cells. The data indicate that the exposure of ciliated respiratory epithelium to mycoplasma cells or membranes results in diminished oxidative metabolism, and that the ability to reduce TTC to its formazan is correlated with relative ciliary activity.

Animals↗
PubMed1995

Susceptibility testing of Candida albicans and Aspergillus species by a simple microtiter menadione-augmented 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay.

We describe a simple microtiter method for determining the susceptibility of Candida albicans and hyphal forms of Aspergillus fumigatus against antifungal agents. The assay measures mitochondrial respiration by determining reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan, a process that is enhanced in the presence of menadione. C. albicans or conidial suspensions of A. fumigatus are seeded into microtiter plates. Hyphal outgrowth of Aspergillus spp. was achieved by a 12 to 14-h culture at 30 degrees C. Antifungal agents (amphotericin B, fluconazole, itraconazole) were added to the cultures for 24 h. Thereafter, incubations were continued for 3 h in the presence of MTT plus 0.1 mM menadione. Formazan formation was quantified photometrically after extraction of the formazan with acid isopropanol. Well-defined dose-response curves reflecting impairment of mitochondrial function by the antifungal agents were obtained. With C. albicans, the results correlated excellently with the MIC determinations performed according to the standard macrodilution procedure. In confirmation of a recent report, it was found that fluconazole was unable to exert its fungistatic action on a sensitive C. albicans strain in the presence of serum. The presented method can easily be integrated in the standard repertoire of a diagnostic microbiology laboratory and should prove useful as a means to assess the antifungal action of various agents on yeasts and filamentous fungi in the presence and absence of serum proteins or body fluids.

Amphotericin B↗
PubMed1987

Linearity in dehydrogenase reaction rate studies in tissue sections is affected by loss of endogenous substrates during the reaction.

We studied the effect of section thickness on the reaction rate of glucose-6-phosphate dehydrogenase (G6PD) activity in unfixed sections of rat liver by use of continuous monitoring by microdensitometry of the reaction product as it formed in the section during incubation. Tetranitro BT or nitro BT was used as final electron acceptor and polyvinyl alcohol as tissue stabilizer. Each test minus control reaction curve deviated from linearity during the first 2 min of incubation. This was mainly due to loss of low molecular weight endogenous dehydrogenase substrates from the surface of the section. For any given reaction, the same absolute amount of endogenous substrate was lost from each section, and hence a much greater proportion was lost from the thinner sections. Such losses lead to a deficit in (nonspecific) formazan production. There was a greater loss from, and hence a greater deficit in, formazan production in sections incubated at 30 degrees C than at 37 degrees C and when nitro BT was used instead of tetranitro BT, but the greatest loss of endogenous substrates occurred in sections incubated in control media. Therefore, greater losses seemed to occur when the reactions were slower because of failure to overcome the critical supersaturation level of the formazan. A consequence of this was a non-linear test minus control response during the first minutes of the incubation.

Animals↗
PubMed1995

The MTT tetrazolium salt assay scrutinized: how to use this assay reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics, IC50-values and cell survival.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay is widely used for in vitro measurement of the metabolic viability of cell cultures subjected to different culture conditions. This convenient assay, which is based on the ability of viable cells to produce formazan, can be affected significantly by a number of conditions. These conditions can be roughly divided into groups, firstly influences which affect the spectrum of the produced formazan and secondly influences which affect the amount of formazan produced per cell. We studied the various chemical and biochemical aspects involved in the MTT assay. Our data indicated that microscopical viewing of cell cultures before and after performing the assay, a medium renewal with a well-defined MTT-incubation medium at the end of the culture period and regular cell counting are essential steps to ensure a reliable performance of the MTT assay. In conclusion, providing the necessary precautions are taken, the MTT assay can be used reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics, IC50-values and cell survival.

Cell Division↗
PubMed1995

Nonmitochondrial oxygen utilization by rabbit blastocysts and surface production of superoxide radicals.

A minimum value for nonmitochondrial oxygen utilization in rabbit blastocysts at day 6 post coitum was determined by measuring oxygen consumption in the presence of cyanide. A microcathode oxygen electrode was used to monitor oxygen concentration continuously during blastocyst incubation in a newly devised culture medium, and the uninhibited blastocyst was found to consume 2.79 +/- 0.09 microliters O2 h-1 cm-2. This rate was reduced by 51% in the presence of 1 mmol KCN l-1. The addition of nitroblue tetrazolium to the cyanide-containing medium reduced net oxygen consumption by an additional 23% as the nitroblue tetrazolium was reduced to formazan. The ability of rabbit blastocysts to reduce nitroblue tetrazolium in the presence of cyanide was investigated using a spectrophotometric assay. Fractionation of blastocyst cells revealed that the enzymatic activity chiefly responsible for formazan production partitioned with the membrane/particulate fraction and could be solubilized by the detergent NP40. The enzyme was NAD(P)H-dependent, did not require divalent cations for activity, and appeared to contain no haeme moiety. The rate of formazan production in the spectrophotometric assay was markedly reduced by the presence of superoxide dismutase. The oxygen electrode and spectrophotometer data indicate that there is a superoxide-generating NAD(P)H oxidase on the blastocyst surface. Calculations based on the average surface area of rabbit blastocysts at day 6 show that these embryos can produce at least 8 nmoles of superoxide per embryo h-1. Potential deciduogenic effects of blastocyst-derived superoxide and its dismutated product, hydrogen peroxide, are discussed.

Animals↗
PubMed1989

The tetrazolium dye assay for rapid in vitro assessment of cytotoxicity.

The intracellular reduction of a tetrazolium salt (MTT) to a purple formazan is an indicator of cell viability. The MTT test represents a simple colorimetric method to determine cytotoxicity. The present paper describes a new method for solubilisation of the formazan crystals, which makes the assay simpler, more rapid and more reproducible. Different cell lines show different kinetics of formazan formation stressing the need for individual calibration curves. The clear-cut discrimination between cells resistant and non-resistant against oxazaphosphorines indicates that the assay has predictive value for in vivo drug sensitivities. It is concluded that the MTT test is a useful addition to anticancer compound screening programs.

Animals↗
PubMed1978

[Use of albuminous standards in the quantitative cytochemistry of dehydrogenases].

A method for preparing albuminous standards with various concentrations of NBT formazans is proposed. Using these standards, the molar extinction coefficients for half and fully reduced formazans of NBT in the region from 450 to 650 nm were calculated. Since the absorption curves of half and fully reduced NBT formazans are identical either in vitro in the standards or enzymatically in tissues, the extinction coefficients calculated on the basis of these standards may be used for densitometrical estimation of cellular dehydrogenase activity.

Animals↗
PubMed1980

[Inflammatory reaction to polyester threads in guinea pigs. III. Reduction of Nitroblue tetrazolium by peritoneal macrophages in vitro].

Peritoneal cells of guinea pigs treated or not by intraperitoneal injection of minced polyester threads have been tested by the NBT test. In phagocytic cells nitroblue tetrazolium (NBT) is reduced to formazan. This is deposited as a dark blue precipitate in the cytoplasm. Changes in reduction capacity have been quantitated by calculation of percentage of macrophages containing formazan deposits, (Tab. 1), and by extraction and photometric evaluation of formazan (Tab. 2). Peritoneal cells incubated with polyester show an increased NBT reduction, which is more apparent with the photometric evaluation. A statistically significant difference in NBT reduction has been found between the peritoneal cells of the guinea pigs treated "in vivo" with polyester and those of the untreated "in vivo" with polyester and those of the untreated animals, whether or not challenged in vitro with polyester threads. These results suggest a change in cell population following polyester treatment.

Animals↗