Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Trypan Blue”

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 937 records · Page 52Linked to original sources
PubMed1994

Superoxide dismutase ameliorates neuronal death from hypoxia in culture.

BACKGROUND AND PURPOSE: Studies showing efficacy with free radical scavengers have been conflicting, and when protection was demonstrated it was attributed to action at the level of the vascular endothelium. The purpose of this study was to test the hypotheses that neuronal free radical formation plays a role in the ischemic cascade and occurs intracellularly and that free radical scavengers, if taken up intracellularly, will protect against hypoxic damage. METHODS: A tissue culture model of hypoxia followed by restoration of oxygen was employed, using both superior cervical ganglia and hippocampal neurons. Cells were exposed to an anoxic atmosphere of 95% N2 and 5% CO2 and examined 2 to 24 hours later after restoration of oxygen. Determination of survival was measured by trypan blue exclusion. Nitroblue tetrazolium stain was used to assess free radical formation. RESULTS: Pretreatment with free superoxide dismutase did not decrease cell death after hypoxia as measured by trypan blue exclusion. However, when superoxide dismutase was taken up intracellularly under depolarizing conditions (55 mmol/L KCl in the medium), cell death was decreased significantly compared with hypoxic controls (28.7 +/- 4.34 versus 40.3 +/- 4.33; P < .03). During hypoxia neurons reduced nitroblue tetrazolium to form the blue precipitate formazan, and the color change was blocked in neurons pretreated with superoxide dismutase in depolarizing medium. Similar findings occurred in both superior cervical ganglia and hippocampal neurons. CONCLUSIONS: These findings provide evidence to support the role of neuronal free radical formation in cell death secondary to hypoxia. In addition, free radical scavengers, if taken up intracellularly, may partially ameliorate their deleterious effect.

Animals↗
PubMed1990

5'-Deiodination in rat hepatocytes: effects of specific flavonoid inhibitors.

5'-Deiodination of T4 or rT3 in the presence of flavonoids was studied in freshly isolated suspended rat hepatocytes. Flavonoids, a novel group of synthetic deiodinase inhibitors, were designed to act as T4 antagonists. 5'-Deiodination of the prohormone T4 to the thyromimetically active T3 is an essential first step in controlling thyroid hormone action. Hepatocytes were incubated with either 2 microM T4 or 2 microM [125I]rT3 as 5'-deiodinase substrates in the absence and presence of inhibitors (0.1-100.0 microM). T3 production from T4 was determined by T3 RIA, and [125I]iodide release from [125I]rT3 was alternatively used as a technically more simple and rapid but sensitive deiodinase assay. Aurones and flavones inhibited both T4 5'-deiodination and rT3 5'-deiodination, with half-maximal inhibitor concentrations from 3-45 microM. Aurones were equally potent in both assays. 3-Methyl-flavones, designed as rT3 analogs, were more active by a factor of 3-5 with T4 5'-deiodination than with rT3 5'-deiodination, with the exception of one relatively cell-toxic compound. Hepatocyte viability was controlled by trypan blue dye exclusion as well as by measuring gluconeogenesis from exogenously added 10 mM lactate. Some of the flavonoids inhibited gluconeogenesis at concentrations that had no effect on trypan blue dye exclusion. Flavonoid inhibitors reduce 5'-deiodinase activity in intact hepatocytes in concentrations equimolar to those of substrates. Therefore, synthetic flavonoids may be suitable substances for further study of iodothyronine physiology or, after modification, could be useful as a new class of antiiodothyronine drugs.

Animals↗
PubMed1978

[The time lapse of the cytostatic effect of ifosfamide].

The time lapse of the effect of ifosfamid on the solid DS carcinosarcoma has been studied using 204 Wistar rats. The main results and the conclusions are as follows: 1. The transplantability of the tumor is abolished two hours after the i.v. application of 180 mg/kg isofamid (cessation of tumor cell proliferation). 2. Yet, the tumor tissue to be grafted is not damaged thoroughly by this treatment. It is possible that the still viable tumor cells were killed by the non-suppressed immune system of the recipients. 3. As determined by trypan blue dye exclusion and registration of glycolytic activity, the main part of tumor cells remains viable. As lately as 4 days after the therapy 80% of the cells incorporate trypan blue and the glycolytic activity is inhibited in the order of 80%. 4. It is to be expected that within two hours, a period sufficient for proliferation inhibition of tumor cells, only 30% of the active form of the drug administered can be found in the tumor. In this context the toxification (activation) kinetics of ifosfamid is discussed and an optimized, programmed infusion is considered. 5. The treatment with ifosfamid does not affect at least up to the third day the tumor hyperacidification attainable by a long-lasting glucose infusion.

Animals↗
PubMed1989

Studies on acute toxic effects to keratinocytes induced by hematoporphyrin derivatives and laser light.

Human epidermal keratinocytes were grown in culture and the uptake of hematoporphyrin derivatives (HPDs) used in photodynamic therapy was estimated. Keratinocytes loaded with HPDs were irradiated with laser light of 632 nm generated by a helium-neon laser and cell toxicity was determined by the trypan blue exclusion test and the measurement of enzyme release. With increasing intracellular concentration of HPDs and with increasing intensity of the laser light, an increasing number of cells took up trypan blue and released the cytosolic enzyme lactate dehydrogenase and the lysosomal enzyme acid phosphatase after 1 h incubation of the irradiated cells at 37 degrees C. Cytotoxicity was less pronounced when the irradiated cells were incubated at 0 degree C indicating the involvement of enzyme reactions in cell death. No lipid peroxidation as measured by malondialdehyde and ethane formation was detectable. Our results suggest that during photodynamic therapy with HPDs and laser light epidermal keratinocytes may be seriously damaged. The data indicate that not lipid peroxidation but rather the activation of lysosomal enzymes is responsible for the cytotoxicity observed.

Acid Phosphatase↗
PubMed2005

Comparison of para-aminophenol cytotoxicity in rat renal epithelial cells and hepatocytes.

Several chemicals, including para-aminophenol (PAP), produce kidney damage in the absence of hepatic damage. Selective nephrotoxicity may be related to the ability of the kidney to reabsorb filtered water, thereby raising the intraluminal concentration of toxicants and exposing tubular epithelial cells to higher concentrations than would be present in other tissues. The present experiments tested the hypothesis that hepatocytes and renal epithelial cells exposed to equivalent concentrations of PAP would be equally susceptible to toxicity. Hepatocytes and renal epithelial cells were prepared by collagenase digestion of tissues obtained from female Sprague-Dawley rats. Toxicity was monitored using trypan blue exclusion, oxygen consumption and ATP content. We measured the rate of PAP clearance and formation of PAP-glutathione conjugate by HPLC. We found that renal epithelial cells accumulated trypan blue and showed declines in oxygen consumption and ATP content at significantly lower concentrations of PAP and at earlier time points than hepatocytes. The half-life of PAP in hepatocyte incubations was significantly shorter (0.71+/-0.07 h) than in renal epithelial cell incubations (1.33+/-0.23 h), suggesting that renal epithelial cells were exposed to PAP for longer time periods than hepatocytes. Renal epithelial cells formed significantly less glutathione conjugates of PAP (PAP-SG) than did hepatocytes, consistent with less efficient detoxification of reactive PAP intermediates by renal epithelial cells. Finally, hepatocytes contained significant more reduced glutathione (NPSH) than did renal epithelial cells, possibly explaining the enhanced formation of PAP-SG by this cell population. In conclusion, our data indicates that renal epithelial cells are intrinsically more susceptible to PAP cytotoxicity than are hepatocytes. This enhanced cytotoxicity may be due to longer exposure to PAP and/or reduced detoxification of reactive intermediates due to lower concentrations of reduced NPSH in renal epithelial cells than in hepatocytes.

Aminophenols↗
PubMed1991

Methods for improving tissue culture of human tracheo-bronchial epithelium obtained at autopsy.

Human tracheo-bronchial epithelium obtained from autopsy, surgery, and organ donation will have areas of both viable and non-viable cells. It is important in the initial establishment of epithelial explant and cell cultures that injured, non-viable mucosal epithelium not be used for the cultures. Autopsy cases selected for culture should initially be chosen on the basis of a shorter post mortem interval and cause of death in order to increase the rate of successful culture. Staining the epithelium with the vital dye, trypan blue, in combination with phase contrast microscopy of the bronchial tissues will further identify those areas of the mucosa that are enriched for viable cells. The dead, non-viable areas are trypan blue positive, while the viable areas are clear and have foci of beating, motile cilia. Treatment of the mucosal tissue with mucolytic agents to remove cell debris, dead cells, and microbes trapped in the mucus material will further improve the chances for successful culture. Human tracheo-bronchial epithelium, although non-sterile and often injured at time zero for numerous reasons, can effectively be used in vitro pathophysiology studies.

Aryl Hydrocarbon Hydroxylases↗
PubMed2000

Specific determination of endothelial cell viability in the whole cell fraction from cryopreserved canine femoral veins using flow cytometry.

Abstract: An efficient method for specifically determining the viability of endothelial cells (EC) from cells dissociated from the human saphenous vein was investigated. Three different methods, trypan blue staining assay, [3H]-proline incorporation assay, and flow cytometry (FCM), combined with the fluorescein isothiocyanate conjugated with Griffonia simplicifolia agglutins (GS1-FITC)/propidium iodide (PI) double staining, were used. Both trypan blue staining and [3H] proline incorporation assays demonstrated less sensitivity to determine viability of EC differentially from the other cells. FITC-GS1 showed prominent binding to the vascular EC and could be counted by FCM including PI on dead cells. Following the cryopreservation process, the GS1-FITC/PI FCM analytical method was adopted to test simultaneously the viability of whole cells and EC from the same tissue, human saphenous veins, and mongrel dogs' femoral veins after harvesting, antibiotic solution treatment, and thawing. The viability of the whole cells from veins decreased with a significant difference (p < 0.05) from that of EC after thawing.

Animals↗
PubMed1993

Comparison of pig hepatocyte isolation using intraoperative perfusion without warm ischemia and isolation of cells from abattoir organs after warm ischemia.

Enzymatic hepatocyte isolation using warm ischemic pig livers from an abattoir was compared with isolation using in situ perfused organs. Using organs from animals of 30 kg body mass (BM), the intraoperative perfusion showed superior results. The use of livers from abattoir pigs of 40-50 kg BM after warm ischemia resulted in a lower yield of hepatocytes and in high rates of injured cells. The mean yield in the intraoperative perfusion group was 68 +/- 11% (wet weight), the maximum yield in the abattoir organ group was 58%. The mean viability in the intraoperative perfusion group was 65 +/- 14% (trypan blue) compared with a maximum viability of 39% in the abattoir liver group. Additional purification by density gradient centrifugation improved the viability of the abattoir liver group to a mean of 95% (trypan blue). The use of pig livers from large abattoir animals required additional purification steps to improve viability since the cell yield is considerably lower than with intraoperative organ perfusion. In general, hepatocyte isolation from abattoir organs is not recommended.

Abattoirs↗
PubMedDate not supplied

Oxalate and calcium oxalate crystals are injurious to renal epithelial cells: results of in vivo and in vitro studies.

Calcium oxalate (CaOx) nephrolithiasis was induced in male Sprague-Dawley rats by administration of 0.75% ethylene glycol. Urinary excretion of lactate dehydrogenase (LDH) was used as a marker of cellular injury. Lipid peroxides (LP), as marker for free radical injury, were measured as malondialdehyde (MDA) in urine and the kidneys. Urinary oxalate (Ox), LDH, LP, CaOx crystals, and renal LP and CaOx crystal deposits were examined on day 0, 5, 30 and 60 of the experiment. There were significant differences between control and experimental rats in all the parameters except LDH which did not show a significant increase after 15 days. Subconfluent cultures of MDCK and LLCPK1 cells were exposed to various concentrations of oxalate and/or 500 fg/ml CaOx crystals. Cell viability was assayed by trypan blue exclusion, cellular injury was determined by measuring LDH in the media, and free radical injury was measured as MDA contents of the cells. On exposure to both Ox and/or CaOx crystals trypan blue exclusion decreased and LDH and MDA increased significantly in both tissue cultures. LLC-PK1 appeared more sensitive. The results indicate that both oxalate and calcium oxalate crystals are injurious to renal epithelial cells in the kidneys as well as in culture.

Animals↗
PubMed2000

Bilirubin- and light induced cell death in a murine lymphoma cell line.

Cells from the mouse lymphoma cell line L5178Y-R were exposed to blue light from phototherapy lamps in the presence of solutions of 160 microM bilirubin supplemented with serum albumin. HPLC analysis showed that the bilirubin solution was photooxidised as a function of increasing light dose. The cells were stained with trypan blue to score necrosis, and apoptosis was assayed by the terminal deoxynucleotide transferase assay (TdT) or by studying the nuclear structure in cells stained with propidium iodide. A rapidly developing apoptosis was observed after light doses killing 60-80% of the cells as judged from the trypan blue exclusion test. The fraction of apoptotic cells was smaller than the fraction of necrotic cells. Exposure of the cells to fractions of light at a high dose rate was compared to the effect of the same total dose at a lower dose rate given as a single fraction. No large differences were found, however, there was a tendency of a higher degree of necrosis as well as apoptosis in the cells receiving the light in fractions at a high dose rate.

Animals↗
PubMed1988

Effect of benzyl chloride on rat hepatocytes.

Benzyl chloride (BCl) is used in the manufacture of basic and acidic dyes, pharmaceutical products, resins, and synthetic tannins. BCl is known to have caused liver malfunctions in some workers exposed to 2 ppm BCl vapors. This study was conducted to investigate the effect of BCl on isolated male rat hepatocytes using several toxicity parameters. The hepatocytes were isolated by a collagenase perfusion technique and were incubated in airtight tubes with 1.8 and 3.6 mM BCl in a shaking water bath at 37 degrees C for 10, 30, 60, and 120 min. Throughout the incubation period the cell viability was determined by trypan blue exclusion and leakage of cytosolic enzymes such as lactate dehydrogenase (LDH), aspartate transaminase (AST), and alanine transaminase (ALT). Exposure to BCl resulted in a significant decrease in cell viability as assessed by trypan blue and significant increase in leakage of these enzymes compared to the controls.

Animals↗
PubMed1977

Cytopathologic effects of chlorhexidine on human cells.

Exposure of human cells in culture to chlorhexidine at concentrations equal to or greater than 0.004% resulted in impaired cellular function and/or cell death. Release of membrane bound 51Cr, inhibition of protein synthesis as measured by incorporation of 3H-leucine into protein-like material, and staining by trypan blue were seen as sequellae to exposure to 0.006% chlorhexidine for 3 hours. Lower doses were capable of inhibiting protein synthesis and releasing 51Cr, but did not result in staining of cells by trypan blue. Exposure of cells to 0.2% chlorhexidine for 30 seconds produced maximal suppression of protein synthesis and release of 51Cr.

Amino Acids↗
PubMedDate not supplied

Cholesterol interferes with the MTT assay in human epithelial-like (A549) and endothelial (HLMVE and HCAE) cells.

Metabolically active cells are able to convert the MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye to blue formazan. This is the basis of the MTT assay, which is among the most widely used screening methods to evaluate cell viability and proliferation. When testing the effects of cholesterol products on the viability of human pulmonary epithelial-like A549 cells using trypan blue staining (cell numbers) and the MTT assay, results were inconsistent. The MTT assay indicated greater than 50% loss of viability with exposure of cells to cholesterol, whereas there was no decrease in viability indicated by trypan blue exclusion and propidium iodide uptake. A similar decrease in MTT reduction was obtained upon cholesterol treatment in human lung microvascular endothelial cells (HLMVECs) and human coronary artery endothelial cells (HCAECs) without loss of viability. This suggested a direct interference of cholesterol with the assay. However, using a cell-free system, there was no decrease in the reduction of MTT by ascorbic acid during incubation with a similar concentration of cholesterol. Light microscopy revealed enhanced exocytosis of formazan granules in presence of cholesterol. Incubation with apolipoprotein A-1 decreased cholesterol-mediated inhibition of MTT assay. These studies indicate decreased MTT reduction as a result of enhanced exocytosis of formazan due to cholesterol. A careful validation of viability assay procedures is therefore suggested in experiments where cholesterol is a constituent, to avoid a potential bias in concluding results of cytotoxicity studies.

Apolipoprotein A-I↗
PubMed2005

[Replication of a model of injury to human renal proximal tubular cells induced by hypoxia/reoxygenation].

OBJECTIVE: To replicate a new model of injury to human renal proximal tubular cells (HK-2) induced by hypoxia/reoxygenation. METHODS: Human renal proximal tubular cell line HK-2 cell was used as the target cell. Tubular cells were divided into six groups: 4 hours of hypoxia, 12 hours of hypoxia, 24 hours of hypoxia, and 24 hours of hypoxia followed by reoxygenation 4, 12 or 24 hours later groups. Each group was accompanied by a control group. Hypoxic culture conditions were produced by covering the culture with liquid paraffin. Trypan blue exclusion was used for cell count and cell viability. The activity of lactate dehydrogenase (LDH) in the culture medium was determined by biochemical method. RESULTS: After being challenged by hypoxia followed by reoxygenation, trypan blue exclusion rate was greater, cell count and cell viability were lower, and the activity of LDH was increased. It indicated that the destruction of integrity of cellular membrane was induced by ischemia/reperfusion injury, and the tubular cells may be injured irreversibly. CONCLUSION: A simple model of hypoxic injury of renal tubular cells is replicated by covering the culture cells with liquid paraffin.

Cell Hypoxia↗
PubMed1994

A six-hour in vitro virulence assay for Listeria monocytogenes using myeloma and hybridoma cells from murine and human sources.

An in vitro cell culture assay using myeloma cells and hybrid lymphocytes was developed which detected pathogenic Listeria strains in just 6 h. Three separate hybridoma cell lines, murine Ped-2E9 and EM-7G1 and human RI.37 and murine myeloma NS1 cells, proved equally sensitive in responding to virulent Listeria species. Listeria monocytogenes along with other Listeria spp., collected from food and clinical sources, were inoculated at 10(8) cfu/ml into a suspension of Ped-2E9 (10(6)/ml). Pathogenic Listeria spp. killed 80% of hybridoma cells by 4 h, as determined by trypan blue exclusion test. Conversely, none of all nonpathogenic Listeria spp. killed the hybridoma cells. Ped-2E9 cells exposed to three strains of L. monocytogenes strains showed 96-97.5% death in 6 h measured by trypan blue staining and release of 91-97% of lactate dehydrogenase (LDH) enzyme. RI.37 cells showed similar results. A multiplicity of exposure (MOE) of 100 L. monocytogenes to 1 hybridoma cell or of 10:1 killed about 80% of the hybridoma cells in 4 or 6 h respectively. The in vitro virulence assay of L. monocytogenes with hybridoma cells compared favorably with the immunocompromised mouse model, yielding results in 6 h instead of 3 days. Intracellular L. monocytogenes and L. innocua were not recovered from Ped-2E9 hybridoma cells after 2 or 4 h of exposure. However, attachment of both L. monocytogenes and L. innocua cells on Ped-2E9 cell surfaces were observed under epifluorescence microscopy. Direct contact of hemolysin positive L. monocytogenes with hybridoma cells is essential to cause death, since hybridoma cells were not killed when they were separated from the growing bacteria by a 0.45 microns filter.

Animals↗
PubMed2006

Study of cytotoxic, genotoxic and DNA-protective effects of selected plant essential oils on human cells cultured in vitro.

OBJECTIVES: To investigate cytotoxic, genotoxic and DNA-protective effects of carvacrol and thymol on human hepatoma HepG2 and colonic Caco-2 cells cultured in vitro. METHODS AND RESULTS: Cytotoxicity testing was performed by the trypan blue exclusion technique. Level of DNA lesions induced in human cells with carvacrol, thymol or their combinations with hydrogen peroxide (H(2)O(2)) were measured by alkaline single cell gel electrophoresis (comet assay). The trypan blue exclusion technique showed that though the metabolically more competent human hepatoma HepG2 cells were more sensitive to the toxic effects of carvacrol or thymol than colonic Caco-2 cells, which contained lower levels of metabolizing enzymes, the observed differences were not very expressive. The comet assay technique showed that at concentrations <IC(50) the compounds studied did not induce DNA strand breaks either in hepatoma HepG2 or in colonic Caco-2 cells. On the other side, 24-h incubation of cells in the presence of carvacrol or thymol led to a significant protection of the cells studied toward DNA damage induced by H(2)O(2). CONCLUSIONS: Based on our results it can be concluded that the cytotoxic effects of selected components of essential plant oils on human hepatoma HepG2 and colonic Caco-2 cells were not associated with a DNA-damaging effect. Carvacrol and thymol reduced the level of DNA lesions induced by H(2)O(2). We assume that the observed DNA-protective effects of these plant volatiles can be associated with their antioxidative activity.

Caco-2 Cells↗
PubMed1979

Sensitive and convenient quantitation of antibody binding to cellular antigens using glutaraldehyde preserved cells.

As a preliminary step in the identification and isolation of antibodies to human cancers, we have developed a sensitive and convenient assay for antibody binding to cellular antigens. The basis for the method is antibody binding to glutaraldehyde-fixed cells (AbGfC) and quantitation with radioiodinated staphylococcal protein A (SpA). Glutaraldehyde fixation of intact cells, which does not appear to effect the ability to form antigen-antibody complexes, provides a convenient and standard supply of target cells which may be stored at 4 degrees C and used in the assay over a period of several months. The amount of antibody specifically bound to the cells is quantitated by the addition of 125I-labeled SpA. The sensitivity of the method was compared with two complement-dependent cytotoxicity methods (trypan blue exclusion and 51Cr release assays) and tested with two antisera to human lung cancer and one antiserum to a membrane antigen of a murine lymphoma. These comparisons indicated much greater sensitivity when compared with the trypan blue exclusion assay and equivalent sensitivity with greater dose response characteristics when compared with the 51Cr release assay.

Aldehydes↗
PubMed1984

Degranulation of mast cells and inhibition of the response to secretory agents by phototoxic compounds and ultraviolet radiation.

The symptoms of cutaneous phototoxicity from coal tar compounds and the nonsteroidal anti-inflammatory drug benoxaprofen are characterized by wheal and flare formation which is mediated by histamine released from dermal mast cells. Rat serosal mast cells were used as an in vitro model system to study the direct effect of phototoxic compounds on mast cell degranulation. The coal tar compounds studied included acridine and pyrene. Combined exposure of cells to acridine and UVA (320 to 400 nm) radiation caused mast cells to degranulate, as assayed by the release of [3H]serotonin. Maximum [3H]serotonin release (70 to 80%) was obtained with 50 microM acridine and 300 kJ/m2 UVA. Pyrene (25 microM), when photoexcited with UVB (280 to 360 nm) radiation, caused about 80% release of [3H]serotonin. No degranulation occurred with 20 microM benoxaprofen and UVB doses up to 7.2 kJ/m2. Trypan blue staining correlated well with degranulation caused by acridine plus UVA; however, with pyrene plus UVB there was greater [3H]serotonin release than dye uptake. Excitation of photosensitizers with doses of UV radiation that did not cause trypan blue staining suppressed degranulation of mast cells in response to chemical stimulation. Acridine, pyrene, and benoxaprofen in the presence of UV radiation inhibited the mast cells from responding to compound 48/80 or the calcium ionophore, chlortetracycline. Two other phototoxic compounds, chlorpromazine and deoxytetracycline, also abolished degranulation by compound 48/80. These findings indicate that phototoxic compounds: (1) cause degranulation in the presence of high doses of UV radiation; and (2) suppress degranulation of mast cells in response to secretory stimuli at doses of UV radiation that do not cause release of mediator.

Animals↗