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At least 667 records · Page 37Linked to original sources
PubMed1985

A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by gamma-interferon.

We describe a simple method by which the insoluble blue formazan dye produced by the reduction of nitro blue tetrazolium can be dissolved without heating using potassium hydroxide and dimethyl sulphoxide. This modification enhances the sensitivity and increases the applications of tests performed using the microELISA method and removes variations caused by uneven cell monolayers. It also allows quantification of NBT reduced by cells adherent to coverslips or in larger wells or Petri dishes, and can be used as a sensitive assay for macrophage activation by gamma-interferon.

Animals↗
PubMed1986

Further studies on the ELISA-spot technique. Its application to particulate antigens and a potential improvement in sensitivity.

Applications of the ELISA-spot technique to particulate antigens (sheep erythrocytes and E. coli) are reported; sheep erythrocytes were used to ensure a rigorous comparison of the spot assay and the haemolytic plaque assay. The spot technique was also applied to soluble antigens (dextran and trinitrophenyl-lipopolysaccharide) to assess the putative occurrence of non-haemolytic antibodies. In most instances, the spot assay disclosed higher numbers of antibody-secreting cells than the plaque assay. An examination of the kinetics of spot formation demonstrated that spot development was most rapid during the first and second hours of enzyme activity and slowed thereafter, although the numbers of spots at 16 h were higher than those at 2 h. To shorten the assay time a redox reaction (which yields an insoluble formazan) was coupled to the enzymatic reaction. In duplicate assays, this improved technique gave larger numbers of spots in a shorter time, than the conventional assay.

Animals↗
PubMed1986

An improved colorimetric assay for interleukin 2.

Mosmann's method for measuring the number of viable cells with a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT), was modified to make it possible to measure a large number of interleukin 2 (IL-2) samples at one time with less labor and more accuracy. Each step of the method was examined in detail and modified (the modified MTT method). An IL-2-dependent mouse natural killer cell line, NKC3, was used as an indicator cell line. The incubation period before adding MTT was reduced to 24 h, A solution of 10% sodium dodecyl sulfate-0.01 N HCl was used to dissolve the MTT formazan produced. We have compared the values obtained by the modified MTT method and the conventional [3H]thymidine method (3H-TdR method), and confirmed that the estimates of IL-2 content were almost equal. The variation of IL-2 content measured by both methods was within 5% in terms of the standard error.

Cells, Cultured↗
PubMed1986

A tetrazolium method for staining peroxidase labels in blotting assays.

A sensitive staining method of horseradish peroxidase-labeled immunoglobulins on nitrocellulose membrane was established by employing a reaction chain leading to formazan formation with phenol as a substrate of peroxidase and NADH as a hydrogen donor to reduce nitro blue tetrazolium. Higher concentrations of NADH relative to phenol were necessary to increase the intensity of staining and to ensure a wide dose-response range of color production with respect to the applied enzyme activities. By an optimized tetrazolium method in combination with antibody-affinity blotting, as low as 4 ng/ml alpha-fetoprotein was detected and 3-4-fold greater color intensities in a working assay range as compared with those of existing methods were obtained. The present technique of peroxidase staining may prove to have a wide application for the enzyme immunoassay using blotting modalities.

Immunoenzyme Techniques↗
PubMed1986

Enzyme amplification for immunoassays. Detection limit of one hundredth of an attomole.

A method is described for increasing the response of enzyme immunoassays employing alkaline phosphatase as the label initiating 2 sequential catalytic reactions. First, NADP is dephosphorylated to produce NAD, which catalytically activates a specific redox-cycle involving the enzymes alcohol dehydrogenase and diaphorase. During each turn of the cycle 1 molecule of a tetrazolium salt is reduced to an intensely coloured formazan. The method is capable of detecting as little as 0.01 amol alkaline phosphatase, and when applied to an immunoassay for TSH a sensitivity (zero + 2.5 standard deviations) of 0.0013 mIU/l was obtained.

Alkaline Phosphatase↗
PubMed1987

Rapid, quantitative microassay for the monokine respiration inhibitory factor.

The murine monokine respiration inhibitory factor (RIF) induces lesions at Complex I and Complex II of the mitochondrial electron transport chain (ETC) of tumor cells; these lesions in the ETC appear closely linked with cytostasis of the targets. In this report we describe the use of the sensitive murine mammary adenocarcinoma line EMT-6 in a colorimetric microassay for the effects of RIF on the ETC and target replication. The participation of cytolytic molecules in this assay system was excluded because of the resistance of the target to their effects. The endpoint for the assay was the ETC-mediated reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to its colored formazan. The two major coupling sites of MTT in the ETC of EMT-6 cells are shown to be proximal to Coenzyme Q, detected either by malate oxidation through Complex I or succinate oxidation through Complex II. The assay was sensitive to both RIF-induced lesions at these dehydrogenases and to the cytostasis-linked reduction in target cell number. The assessment of ETC lesions by this microassay correlated directly with that determined by the less sensitive polarimetric assay based on oxygen consumption. We demonstrate the application of this microassay to parameters for the production of RIF by activated murine macrophages, and to initial molecular characterization of this mediator.

Animals↗
PubMed1990

A sensitive two-site sandwich enzyme immunoassay for human angiotensin converting enzyme utilizing monoclonal antibodies.

A sensitive enzyme immunoassay was developed for human angiotensin converting enzyme. Monoclonal antibodies specific for two unique converting enzyme epitopes were utilized to develop a two-site sandwich enzyme immunoassay. Alkaline phosphatase conjugated to the detecting antibody hydrolyzes nicotinamide adenine dinucleotide phosphate (NADP) to NAD. Subsequently, NAD is cycled between its reduced and oxidized forms by an alcohol dehydrogenase/diaphorase catalyzed redox cycle. Each cycle converts iodonitrotetrazolium violet to a highly colored formazan which is quantitated. With this assay, as little as 94 pg/ml of native converting enzyme is detectable without interference from either therapeutic or endogenous converting enzyme inhibitors.

Animals↗
PubMed1990

A simple and rapid method to determine hematopoietic growth factor activity.

A rapid and simple colorimetric microassay method for the determination of hematopoietic growth factor activities was established. The assay was used to detect CSF-1, GM-CSF, and IL-3 activities. The assay was based on the metabolism of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by metabolically active cells. Results obtained with the colorimetric microassay are comparable with those obtained with the soft agarose assay. Advantages of the colorimetric microassay include the conservation of reagents, the shorter incubation time for the experiment, the shorter assay time, and the ability to evaluate large numbers of samples.

Animals↗
PubMed1993

Simple colorimetric cell-cell adhesion assay using MTT-stained leukemia cells.

A new and simple method for quantitating adhesion of leukemia cells, HL60, to endothelial cells was developed. HL60 cells were incubated with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) which forms a blue dye of formazan in the cells. MTT did not inhibit shape change of HL60 cells induced on activated endothelial cells at 90 min. The expression of a cell adhesion molecule, LFA-1, in HL60 cells at 21 h was not inhibited by MTT. After incubation of the MTT-labeled cells and endothelial cells, non-adhered cells were washed out. Adhered cells were lysed with dimethylsulfoxide, and quantitated by measuring absorbance at 540 nm. The absorbance was well correlated with the adherent cell numbers measured by direct counting or by 51Cr-labeling method. U937 and Ramos cells were also quantitatively labeled by MTT. The method has the advantage of being easy, simple and applicable to various cell-cell adhesion assays.

Cell Adhesion↗
PubMed1994

A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia.

The MTT-colorimetric monocyte mediated cytotoxicity assay, based upon the ability of living cells to reduce 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) into formazan, was evaluated using leukemic cells from five representative human leukemic cell lines and from 28 patients with acute myeloid leukemia (AML). An excellent linearity between absorbance and leukemic cell number was observed up to 5 x 10(4) cells/well and 50 x 10(4) cells/well for all cell lines and patients samples tested, respectively, in a 96-wells microtiter culture system. A huge variability in the susceptibility of leukemic cells to purified and IFN-gamma-activated human monocytes could be observed at effector-to-target cell (E:T) ratios of 1. The mean signal-to-noise ratio of the MTT assay for monocyte-leukemic cell mixtures from patients was 2.69 +/- 0.39 at E:T 1. In conclusion, the MTT based monocyte mediated cytotoxicity assay should be useful for studying the susceptibility of a variety of leukemic cells from cell lines and from patients with AML to monocytes in a rapid, sensitive and semi-automated manner.

Acute Disease↗
PubMed1994

An improved MTT assay using the electron-coupling agent menadione.

A modification of the MTT based tetrazolium colorimetric assay is described. Using the electron-coupling agent menadione formazan formation by murine splenocytes and P-815 cells was significantly increased whereas dye reduction by macrophages was hardly influenced. These observations suggest that it should be possible to improve the tetrazolium based cytotoxicity assays of murine macrophages against cells of the syngeneic tumour cell line P-815.

Animals↗
PubMed1995

Comparison of [3H]thymidine incorporation with MTT- and MTS-based bioassays for human and murine IL-2 and IL-4 analysis. Tetrazolium assays provide markedly enhanced sensitivity.

The sensitivity of [3H]thymidine incorporation and MTT/MTS colorimetric bioassays for detection and quantitation of murine and human IL-4 and IL-2 were compared in CT.4S, CT.h4S and HT-2 bioassays respectively. We reasoned that low levels of cytokine, insufficient to induce cell proliferation (thus, DNA synthesis and [3H]thymidine incorporation), may be sufficient to maintain the viability of the bioassay cells in culture. Because colorimetric assays such as those employing MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) or MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-( 4-sulfonyl)-2H- tetrazolium) measure conversion of these salts to intensely colored formazan products by mitochondrial enzymes independent of whether proliferation is induced, we reasoned that such assays could be superior for detection of low levels of cytokine protein. Direct comparison of these approaches demonstrated that the MTT- and MTS-based assays were consistently able to detect 2-16-fold lower cytokine levels than methods based on [3H]thymidine incorporation. Moreover, the MTT and MTS assays exhibited higher precision with standard deviations of < 1-4% vs. 5-15% for thymidine incorporation. This finding is of particular importance in approaches such as limiting dilution analysis, or primary bulk culture of antigen-stimulated human lymphocytes, where levels of cytokine production may be extremely low.

Animals↗
PubMed1991

Antineoplastic ether-linked phospholipid induces differentiation of acute myelogenous leukemic KG-1 cells into macrophage-like cells.

The culture of a human acute myelogenous leukemic cell line (KG-1) with a synthetic ether-linked phospholipid: 1-0-octadecyl-2-0-methylglycerol phosphocholine (ET-18-OCH3), suppressed the growth of the KG-1 cells while the variant subline, (KG-1a cells) similarly treated was unresponsive. The growth inhibition of the KG-1 cells was accompanied by morphological changes into cells of the monocyte/macrophage lineage. Histochemically, the ET-18-OCH3-treated KG-1 cells increase 17-fold in the nonspecific esterase activity when compared to control non-treated cells, whereas they responded negatively in the assay for the reduction of soluble nitroblue tetrazolium into insoluble blue formazan deposits (a marker for cells of the granulocytic lineage). Taken together, our data revealed that the synthetic ether-lipid inhibited the growth of the KG-1 acute myelogenous leukemic cells while inducing the differentiation of these cells into cells of the monocyte/macrophage-lineage. These effects of the synthetic ether lipid raise the possibility that naturally occurring ether-linked phospholipids may likewise function in vivo to modulate hyperproliferative processes and thus warrant further explorations.

Antineoplastic Agents↗
PubMedDate not supplied

Onchocerca gutturosa and O. volvulus: studies on the viability and drug responses of cryopreserved adult worms in vitro.

The viability and drug responses of cryopreserved adult Onchocerca have been examined in vitro. Male worms were cryopreserved in liquid nitrogen (-196 degrees C) using ethanediol as a cryoprotectant in a 2-step incubation procedure. After thawing, 85-90% of O. gutturosa males were normally motile. These motile worms were evaluated for viability using 4 measurements (long-term motility/survival in culture; [U-14C]adenine uptake and leakage; glucose utilization; MTT-formazan colorimetry) and were no different from unfrozen controls. Subsequent experiments demonstrated that the motility responses of cryopreserved worms exposed to the antifilarial drugs ivermectin, CGP 6140 and levamisole were virtually identical to unfrozen controls. Some success was also obtained with this technique in cryopreserving O. volvulus males, with 2 thawed specimens surviving in culture for 93 and 106 d respectively. Following collagenase isolation, female worms were cryopreserved in medium +10% serum without protectant at -79 degrees C. A batch of 8 female O. gutturosa were all motile when thawed 14 d later, with a mean survival time (based on 5 specimens) of 71 d (range 60-90). However, a batch of worms transferred from -79 degrees C to -196 degrees C were badly damaged when thawed. Female O. volvulus were cryopreserved at -79 degrees C in Guatemala and sent by air freight on solid CO2 to the UK. Most specimens were active when thawed. Survival of motile specimens ranged from 7 to 272 d in culture. It is concluded that these techniques are of practical value for the storage and transportation of adult Onchocerca.

Adenine↗
PubMedDate not supplied

The effects of ivermectin used in combination with other known antiparasitic drugs on adult Onchocerca gutturosa and O. volvulus in vitro.

The effects of ivermectin at a concentration of 3.13 x 10(-6) M used in combination with other antiparasitic drugs on the viability of adult Onchocerca in vitro were assessed using MTT colorimetry and worm motility levels. When ivermectin was used against male O. gutturosa over a 7 d period in combination with suramin (5 x 10(-5) M), CGP 6140 (3.13 x 10(-6) M), CGP 20376 (1.95 x 10(-7) M), mefloquine (3.13 x 10(-6) M), levamisole (3.13 x 10(-6) M), mebendazole (5 x 10(-5) M), flubendazole (5 x 10(-5) M) and albendazole (5 x 10(-5) M), there was either no increased effect or only a marginally increased effect on motility levels when compared with the use of ivermectin alone. MTT colorimetry revealed that in most cases there was a cumulative effect of the 2 drugs used in combination but not a synergistic effect. In a trial extended to 26 d it was demonstrated that the combination of ivermectin and suramin did not produce a greater inhibition of motility than ivermectin alone. Using female O. volvulus, the activity of ivermectin, CGP 6140 and the 2 drugs combined was examined. The motility of all 3 groups exposed to drug(s) was suppressed by 24 h compared with controls. MTT colorimetry performed on day 7, using the pre-weighed anterior end of each worm, illustrated that ivermectin alone produced a 43.4% inhibition of formazan formation compared with controls, CGP 6140 alone produced 50.6% inhibition, while the drug combination produced a 72% inhibition, equivalent to the heat-killed control.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Histochemical localization of formaldehyde dehydrogenase in the rat.

Formaldehyde dehydrogenase (FDH) activity has been demonstrated biochemically in the olfactory and respiratory mucosae and in the liver of the rat, but the cellular localization of this enzyme has not been investigated. A histochemical procedure was developed to permit cellular localization of FDH. This allowed us to examine the relationship between distribution of FDH and formaldehyde-induced toxicity. Cold-processed glycol methacrylate embedded tissues were used to localize FDH activity in the rat respiratory tract, kidney, liver, and brain. Five- or ten-micrometer tissue sections were incubated in a reaction mixture containing formaldehyde (HCHO), glutathione (GSH), NAD+, nitroblue tetrazolium, pyrazole, and disulfiram. A blue formazan precipitate was formed at the site of FDH activity. Epithelial cell cytoplasm of both the respiratory and the olfactory mucosae of the nose stained for FDH, and olfactory sensory cell nuclei were also positive. Underlying Bowman's and seromucous glands were weakly positive. The lung had FDH activity located mainly in the Clara cells of the airways, with only diffuse weak activity in the lung parenchyma. Liver had activity in the cytoplasm of the hepatocytes, while in the kidney FDH was most prominent in the brush border of the P2 segment of the proximal tubules. Brain white matter stained strongly for FDH, while in gray matter only the neuropil exhibited weak activity. Corresponding tissue sections were stained for sulfhydryls; these sections indicated that GSH is likely to be present in all cells with FDH activity. For the respiratory tract these results demonstrate distinct differences between the location of FDH activity and previously reported nonspecific aldehyde dehydrogenase activity in the nose (M. S. Bogdanffy, H. W. Randall, and K. T. Morgan, 1986, Toxicol. Appl. Pharmacol. 82, 560-567). While high aldehyde dehydrogenase activities were found in tissues with low toxicities due to acetaldehyde exposure and vice versa, FDH activity was observed in tissues whether or not they exhibited a toxic response to inhaled HCHO. While not able to account for the localized toxicity of HCHO, the presence of FDH and glutathione in the epithelial layer of the nasal cavity presents a barrier to inhaled formaldehyde at low concentrations and may partially explain the observed nonlinearity of HCHO toxicity.

Administration, Inhalation↗
PubMed1988

Cytotoxic effect of T-2 mycotoxin on cells in culture as determined by a rapid colorimetric bioassay.

We developed a colorimetric assay for determining metabolic activity (viability) of cells exposed to toxic agents. This system is based on the ability of mitochondrial enzymes in viable cells to modify a tetrazolium salt into a blue formazan product that can be detected spectrophotometrically at 570 nm. The assay works equally well for mammalian and insect cell lines and at 48 hr color formation is linear over a cell input range of 1.56-50 X 10(4) cells/ml. The inhibitory effects of T-2 mycotoxin on tetrazolium cleavage in L929 cells is comparable to that observed for protein and DNA synthesis (50% inhibition = 6-8 ng/ml). Using this system to analyze the lethal effect of T-2 toxin on cells from various animal species, it was found that bovine cells were the most sensitive (50% inhibition at 2.2 ng/ml) while hamster cells were the most resistant (50% inhibition at 26.2 ng/ml). Murine cells exhibited intermediate sensitivity (50% inhibition at 10.9 ng/ml). Variable toxin susceptibility was also observed among different cell types. Lymphocytes were 3-fold more sensitive to the T-2 inhibitory effects than comparable tissue culture cell lines. These data indicate that the colorimetric assay system could have broad applications in toxicological studies. Further, the observed differences in species sensitivity may provide insight into the primary mechanism of the T-2 toxin-cell interaction that ultimately leads to cell death.

Animals↗
PubMed1993

Toxic effects of human pancreatic and snake and bee venom phospholipases A2 on MCF-7 cells in culture.

The toxicity of phospholipase A2 (PLA2) has been suggested to be involved in the pathology of a number of severe diseases including septic shock and acute pancreatitis. However, testing the toxicity of these substances is difficult in vivo. In the present study we compared the toxicity of PLA2s from three snake venoms, bee venom and human pancreas on MCF-7 cells grown in culture. Tetrazolium microculture assays were developed to test the cytostatic and cytotoxic effects of PLA2 on MCF-7 cells. These tests are based on the ability of viable cells to reduce a tetrazolium-based compound MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to a blue formazan product. Leakage of lactate dehydrogenase (LD) from the cells into the culture medium was also measured. There were marked differences in the toxicity of the PLA2s tested. Cobra (Naja mosambique mosambique) venom PLA2 was toxic to the cells at a concentration of 4.5 U/ml. Light microscopic changes were seen in the injured cells after 3 hr treatment. Sixty-seven per cent of cells were dead after 24 hr treatment. Treatment for 4 hr caused irreversible changes in the cells. Leakage of LD was noted from 4 hr onwards. Other snake (Crotalus adamanteus and Laticauda semifasciata) venom PLA2s, even after continuous exposure to 4.5 U/ml caused only slight decreases in values obtained in the MTT test. No morphologic changes suggesting a cytotoxic effect were seen. PLA2 from bee (Apis mellifera) venom had no toxic effect, either. Continuous exposure of cells to human pancreatic PLA2 caused a 15% decrease in the MTT-test.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗