Interference of Niagara blue 2B with the teratogenic action of trypan blue.
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Rapid selection of viable transplantable human fetal pancreatic islets by trypan blue exclusion.
A rapid method of separating viable from nonviable human fetal pancreatic islets prior to transplantation is needed to quantify grafted tissue and optimize the possibility of successful treatment of diabetes mellitus in the recipient. After incubation with 0.04% trypan blue in isotonic Krebs-Ringer buffer solution for 15 min, the percentage of islets that excluded trypan blue was found to correlate well with fractional stimulated insulin secretion rates. The incubation procedure did not alter the subsequent insulin secretory capacity of the islets. Trypan blue exclusion rapidly and reliably identifies viable functional islet tissue prior to transplantation.
Cell Separation↗
Ultrastructural changes of the early visceral yolk sac layer of mouse embryos after maternal injection of trypan blue.
The ultrastructural features of the early visceral yolk sac epithelium of normal mouse embryos on day 9 were compared to those whose mothers had received a single subcutaneous injection of 100mg/kg trypan blue on day 8. The following results were obtained: In normal embryos the visceral yolk sac cells are predominantly characterized by numerous membrane bounded inclusions localized in the supranuclear cytoplasm. In embryos of animals treated with trypan, blue, at about 12h after injection large single and only partly membrane bounded vacuoles are observed occupying most of the apical cytoplasm. Up to 24h after injection large cytoplasmic areas are seen which are in a stage of autodigestion possibly due to leakage of the vacuolar content. These alterations are exclusively limited to the visceral yolk sac epithelium whereas in the cells of the embryonic part, e.g. head process, no changes could be found. The observations are discussed in relation to the mechanism of the teratogenic activity of trypan blue.
Animals↗
Trypan blue uptake as a new method to investigate hepatotoxicity in periportal and pericentral regions of the liver lobule: studies with allyl alcohol in the perfused liver.
A new method based on trypan blue uptake was developed to study zonal hepatotoxicity in specific regions of the liver lobule in perfused livers. Allyl alcohol (350 microM), a hepatotoxin that causes necrosis of periportal regions, was infused into livers from phenobarbital-treated rats for 60 min. The subsequent infusion of trypan blue (0.2 mM) stained nuclei in periportal but not midzonal or pericentral regions of the liver lobule. With this new method of dye uptake, comparison of the temporal relationship between metabolic changes and the onset of cellular damage was possible. During the first 10 min of allyl alcohol infusion, allyl alcohol was taken up at rates of 64 mumol/g/h concomitant with an increase in oxygen uptake of 18 mumol/g/h. Allyl alcohol infusion also increased bile flow from 69 to 167 microliters/min and release of oxidized glutathione from 127 to 217 nmol/g/h, respectively. However, during the next 20-min phase of exposure to allyl alcohol, oxygen uptake was inhibited by 60% and allyl alcohol uptake declined by 80%. Bile flow and release of oxidized glutathione also declined continuously between 10 and 30 min and by 30 min stopped completely. Concomitant with the inhibition of oxygen uptake, rates of glycogenolysis and glycolysis increased by 130 and 85%, respectively. Within minutes after maximal inhibition of oxygen uptake, between 30 and 40 min after initiation of allyl alcohol infusion, trypan blue was taken up by hepatocytes in periportal zones of the liver lobule. Subsequently, lactate dehydrogenase began to appear in the effluent perfusate.(ABSTRACT TRUNCATED AT 250 WORDS)
1-Propanol↗
Use of trypan blue for identifying early proximal convoluted tubules.
Proximal convoluted renal tubules (PCT) are difficult to dissect for in vitro study, particularly in newborn animals, and are identified with certainty only when obtained in continuity with their parent glomeruli. We show here that the supravital colloidal dye trypan blue preferentially stains early PCT segments of rabbit in vivo and greatly facilitates their isolation. We also show that trypan blue administration does not adversely affect animal growth rate, kidney weight, or several general measures of kidney function. Using isolated PCT from newborn to adult rabbits, control (unstained) and trypan blue-stained tubules also are found not to differ in lateral membrane surface density, in their ability to maintain normal cell volume or to respond to hypotonic osmotic challenge, and in their swelling rate in isotonic medium after ouabain treatment. We conclude that trypan blue has no adverse effects on PCT cell function while greatly facilitating their isolation for physiological studies.
Animals↗
Medium containing trypan blue and antibiotics for the detection of Cryptococcus neoformans in clinical samples.
A medium containing trypan blue, gentamicin, and chloramphenicol is introduced for the detection of Cryptococcus neoformans and Cryptococcus species from clinical samples. Ten recently isolated strains of Cryptococcus species as well as several clinical isolates of C. neoformans incorporated trypan blue and produced dark blue colonies on this mycological medium, whereas other common yeasts were light blue. The laboratory diagnosis of two cases of cryptococcosis was accomplished by the isolation of C. neoformans on the antibiotic-dye-containing medium. Compared to conventional media supporting large numbers of Pseudomonas aeruginosa and other gram-negative bacilli, the new medium was selective for yeasts. In one instance, the colonization of the respiratory tract by C. neoformans which led to fungemia was traced by the use of the antibiotic-dye medium. The antibiotic mixture, utilized herein, was more effective in suppressing bacteria contained in samples from patients than a medium containing cycloheximide and chloramphenicol.
Candida albicans↗
[Differential trypan blue staining of tumor cells for the determination of apoptosis].
The present paper suggests that trypan blue should be used for differential staining of tumor cells in order to determine apoptosis. The proposed method makes it possible to simplify operations, to reduce the time required to carry out a test, and to enhance the precision of determining the course of apoptosis during routine investigations. The paper also gives data of the comparative studies of the content of apoptotic cells in experimental melanoma B-16 though differential trypan blue staining and classical morphostructural analysis. The difference in the values obtained by these two methods is shown to be 35%.
Apoptosis↗
Comparison of trypan blue dye exclusion and fluorometric assays for mammalian cell viability determinations.
A hemocytometer-based trypan blue dye exclusion cell quantitation and viability assay was compared with a similar assay using simultaneous fluorometric staining with fluorescein diacetate and propidium iodide. Viable and nonviable cell densities were measured, and culture viability was calculated both during the normal growth cycle of a murine hybridoma and in response to the application of millimolar concentrations of either tert-butyl hydroperoxide or ferrous iron. During the early phase of rapid hybridoma cell growth, assay-based differences in viable cell density were not significant. As the culture aged, the trypan blue dye exclusion assay significantly overestimated cell viability, thereby underestimating nonviable cell density and yielding an erroneous estimation of the overall viability of the culture. Because of its lack of ambiguity in the identification of stained, nonviable cells and its resulting increased accuracy in the estimation of culture viability, the fluorometric assay was considered a better choice for the evaluation of cell viability.
Animals↗
Trypan blue teratogenesis in the rat: further observations in vitro.
Explanted 9 1/2- and 10 1/2-day rat conceptuses were cultured for 24 or 48 hours and 24 hours respectively in immediately centrifuged, heat-inactivated rat serum to which trypan blue was added at concentration of 150 micrograms/ml or 300 micrograms/ml or 450 micrograms/ml. The embryos were assessed for normal growth and differentiation using the following criteria: heart beat, vitelline circulation, fusion of the allantois with the chorion, normal turning, normally closed neural tube, presence of optic vesicles, presence of forelimb buds, tail, somites, somite number, yolk sac diameter, crown-rump length, and protein content. The results indicated that trypan blue is more teratogenic when 9 1/2-day conceptuses are cultured for the first 24 hours, and that its teratogenicity decreases after 10 1/2 days. Morphological abnormalities produced included neural tube defects, turning defects, and tail defects, the tail abnormalities being manifest as fluid-filled blebs. Ten and one-half-day conceptuses cultured for 24 hours in treated serum produced predominantly tail defects. The conceptuses were cultured at 11 days, when the embryo lies completely within the yolk sac. Culture of such conceptuses in serum containing 450 micrograms/ml trypan blue produced mainly tail defects. Injection of 0.5 microliters of 0.5% dye solution into the yolk sac cavity also produced tail abnormalities; sham treatment, or the injection of sterile water or a nonteratogenic dye, azo blue, did not affect embryonic development.
Animals↗
Effects of trypan blue on thyroid secretion. Inhibition of purified cathepsin D from bovine thyroid.
Previous studies have demonstrated that trypan blue directly inhibits thyroid secretion when the dye is administered in vitro or in vivo. To further study the mechanisms of inhibition, cathepsin D (EC 3.4.23.5) (thyroidal acid proteinase) has been purified from bovine thyroid. Trypan blue inhibited the proteolysis of both 125I-labeled thyroglobulin and 125I-labeled hemoglobin in both crude lysosomal enzyme preparation and purified endopeptidase and the inhibition was competitive. Inhibition was also observed when the dye was allowed to prebind to either purified enzyme or purified substrate. Inhibition of cathepsin D is shown to account for part of the inhibition of thyroid secretion.
Animals↗
Experimental modification of vitellogenesis in Japanese quail by trypan blue in vivo.
Ovaries of adult Japanese quails were exposed in vivo to the acid bisazo dye trypan blue (TB) which binds to plasma albumin, the plasma precursor of the yolk protein alpha-livetin. By a combination of fluorescence microscopy and electron microscopy alpha-livetin could be localized in the subdroplets of intermediary and yellow yolk spheres. Trypan blue alters vitellogenesis in the non-disc region of follicles in rapid growth in a reversible and dose-dependent way. Less yolk is produced over 24 h and its morphology is different when compared to controls. This yolk is similar to yolk of the germinal disc region where vitellogenesis is known to be inhibited physiologically. Several ultrastructural features of the germinal disc region are also found in the non-disc region of TB-exposed follicles. Our results suggest that the morphology of yellow yolk is linked to the rate of deposition. We propose that the inhibitory action of TB on vitellogenesis can be explained by a defective receptor-ligand dissociation in endosomes.
Animals↗
Side effects of the trypanocidal chemotherapeutics trypan blue and suramin.
Both the uptake of C3-opsonized and of nonopsonized bakers' yeast is inhibited by 1 mM trypan blue. This effect may be explained by inactivation of the C3bi-receptor. But trypan blue also inhibited the uptake of other non-opsonized particles lacking specially binding domains for this receptor. - Suramin has antiphagocytic properties at 1 mM concentration which were explained by its anticomplementary activity. However, the uptake of nonopsonized particles is also inhibited by suramin. The importance of these effects for therapeutic utilization is discussed.
Animals↗
[Trypan blue staining in treatment of macular hole].
The purpose of the study was to estimate the results of macular hole surgery usingpars plana vitrectomy, ILM peeling with and without dye-trypan blue and endotamponade. We observed that trypan blue increases the precision and safety of surgery and improves the postoperative anatomical and functional results.
Adult↗
Combined trypan blue and glyoxylic acid-induced catecholamine fluorescence in the female rat urinary bladder after distension.
The effect of short-term urinary bladder distension on bladder perfusion and adrenergic innervation was studied in Sprague-Dawley rats. Distension was induced for three hours by forced diuresis and balloon obstruction. A 2% solution of trypan blue was injected into the tail vein 12, 24, 36 and 48 hours after distension and the animals were killed 5-10 min after the injection. Whole thick biopsies were taken from the dome, anterior body and base. A combination of trypan blue and catecholamine fluorescence (GIF method) was used to correlate the distension induced changes in blood vessel perfusion and permeability with changes in adrenergic innervation. Both ischaemic damage and adrenergic hypoinnervation were observed after distension. Later marked extravasation of trypan blue was observed in the whole bladder after 12 hours and after 24 hours distension the dome was almost necrotic in appearance and highly ischaemic. After 36 hours a few exploded small, intensely fluorescent cells (SIF cells) were found to be scattered along the blood vessels, and degranulated mast cells had invaded the anterior body and dome. The adrenergic hypoinnervation reached its maximum 48 hours after distension. The hypoinnervation and possibly also the damage to SIF cells, would seem to be related to ischaemia during distension, probably attributable in turn to overstretching of the organ and its blood vessels. This may provide an explanation for the prolonged micturition problems found after bladder overdistension.
Adrenergic Fibers↗
[Trypan blue and alizarin red S sequential staining with subsequent fixation. A modified technic for the study of the corneal endothelium].
A sequential corneal endothelium staining technique with trypan blue (TB)--alizarin red S (ARS)--ethanol-trypan blue is described which can be performed in a few minutes. The first step permits the TB exclusion to be determined as a criterion of normal endothelial cell function. The two following steps permit intercellular staining by ARS and nuclear staining by TB. After each staining procedure, fixation can be performed by methanol or ethanol, both with full-thickness corneal preparations or endothelial flat mounts. In flat preparations, the focussing problems and optical artifacts of full-thickness mounts can be avoided. These modifications render simple sequential staining with subsequent fixation a complement and alternative to specular microscopy and to the customary complicated silver staining of corneal endothelium.
Animals↗
Ultrastructural and cytochemical effects of trypan blue on TSH stimulation of thyroid follicular cells.
Ultrastructural and cytochemical techniques were used to study the effects of trypan blue on the response of mouse-thyroid cells to exogenous stimulation by thyroid stimulating hormone (TSH). The dye delayed the response to TSH resulting in decreased colloid-droplet formation in the apical region of the cells. The dye did not stop the shift of trimetaphosphatase activity from lysosomes to phagolysosomes. The duration of the TSH-induced response was shorter in the dye treated thyroids. Small vesicles, with trimetaphosphatase reaction product, were found near Golgi elements, phagolysosomes, and the plasma membrane facing rhe intercellular space of adjacent follicle cells. Their enzyme activity was not affected by exposure to the dye. These data indicate that the primary effect of trypan blue on the response of thyroid follicle cells to TSH stimulation was reduced endocytosis in the apical region resulting in fewer colloid droplets.
Acid Anhydride Hydrolases↗
Methods in laboratory investigation. Exclusion of trypan blue from microcarriers by endothelial cells: an in vitro barrier function test.
The introduction of microcarriers in cell culture work has offered new possibilities not least in the study of endothelial biology. This article describes the use of microcarriers for selection of endothelial cells from mixed cultures by using a simple migration-transfer technique. Colonies of either endothelial or smooth muscle cells were allowed to form on gelatin-coated dishes with mitomycin-treated feeder cells. Microcarriers were then seeded so that one or two of them attached to each colony. The cells populated the microspheres within 2 to 3 days. Microcarriers from pure endothelial cell colonies were selected for transfer and serial subcultivation. Furthermore, a method to study the barrier function of confluent microcarrier cultures of endothelium was outlined. This method utilizes the fact that intact confluent cells exclude the dye trypan blue from the matrix of the microspheres which otherwise binds the dye tightly. The rate of absorption of trypan blue from a standard buffer containing 0.2% dye and 1 mg/ml of serum albumin by a standard volume of packed cell covered microspheres was determined. It was shown that confluent endothelial cells provided a barrier for the dye for several hours and that the subcellular matrix contributed to the barrier but only to a small extent. Treatment of the cells with dicloxacilline, 0.1 gm/ml, and homocysteine, at 8 mM, also influenced the barrier markedly. Dicloxacilline caused a complete breakdown and homocysteine clearly increased the rate of absorption of the trypan blue. It is suggested that the barrier function of cultured cells may be studied by this technique or future modifications based on similar principles.
Absorption↗
Photometric recording of cell viability using trypan blue in perfused cell cultures.
A perfusion system was developed to increase the reliability of cell viability estimations by continuous measurement of the uptake of trypan blue dye. Monolayer cell cultures were perfused with buffer containing toxic substances and trypan blue, and the staining of cells was continuously recorded at 591 nm in a spectrophotometer. Using mercuric chloride and methylmercuric chloride as test substances with C6 rat glioma cells, time- and dose-dependent increases in light absorbance were obtained over a 12h recording period. Methylmercuric chloride at 10(-6) M caused a half-maximal increase in relative absorbance in 4.5 h, whereas the corresponding time for mercuric chloride was 10.5 h.
Animals↗