Vital dye

Dyes. Vital dyes have been used for many years in a variety of clinical situations. Patent blue V (also called E131, Acid blue 3, Disulfine blue) and Isosulfan blue (also called Patent blue violet or Lymphazurine), belong to the group of triarylmethane dyes and are the most commonly used [24]. Reports of IgE-dependent anaphylaxis arise. Anaphylactic reactions involving methylene blue seems to be very rare, however, several reports of sensitization to both Patent blue and methylene blue have previously been reported. 

Mennel, S. et al.. Patent blue a novel vital dye in vitreoretinal surgery, Ophthalmo-logica, 220,190, 2006. 

Pfutzner, W. et al.. Intraoperative labeling of sentinel lymph nodes with a combination of vital dye and radionuclide tracer results in sentinel lymph node-positive patients, J. Deutsch. Dermatol. Ges., 4, 229, 2006. 

Serbedzija, G. N., Fraser, S. E., and Bronner-Fraser, M. (1990). Pathways of trunk neural crest cell migration in the mouse embryo as revealed by vital dye labelling. Development 108 605-612. 

MTX interferes with the growth of cancer cells by inhibiting the metabolism of folic acid. Drug efficacy was evaluated in vitro by MTT assay, as described above, and by Trypan Blue exclusion. Trypan Blue is a non-vital dye excluded by viable cells, but selectively staining dead cells. According to Figure 13.7, higher suppression of cell... 

The test is based on an in vitro assay of the uptake of the dye, neutral red (NR), in Balb/c 3T3 fibroblasts. It was developed to detect the phototoxicity induced by the combined interaction of the test substance and light of the wavelength range from 315 to 400 nm, the so-called UVA. The cytotoxicity is evaluated in the presence (+UVA) or absence (-UVA) of UVA light exposure, after application of a nontoxic dose of the compound. The cytotoxicological impact is assessed via the inhibition of the fibroblasts to take up the vital dye NR (NR is a weak cationic dye, penetrating easily into the cell membrane by a nonionic diffusion and accumulates in the lysosomes) one day after the initial treatment. Normally, healthy cells may incorporate and bind NR. Alterations of the cell surface or the lysosomal membranes, however, lead to a decreased uptake and binding of the dye. 

Examining of viability of cryopreserved cells of specific phenotype by parallel determining with vital dye and analysis with flow cytometer has demonstrated that CD 13 3 and CD34 cell candidates occurred to be more sensitive to programmed cryopreservation in comparison with more differentiated erythroid precursors (glycohporin-A -positive cells). Gly-A cells viability made 93.75 1.60%. CD 34 cell viability was 79.57 4.13%,... 

At the physiological level it is well established that vital dyes such as nile blue, neutral red and methylene blue retard larval development under normal lighting conditions (12L/12D with source unspecified) (25 27). Female but not male pupal weights are also reduced. Unfortunately experiments were conducted without dark controls so that it is difficult to evaluate the role of photosensitization in these effects. As house flies and fire ants succumb to photosensitization, they lose motor control and become more excitable (28). This suggested a neurotoxic effect and investigation of fire ant acetylcholinesterase vitro revealed that this enzyme was sensitive to photo-oxidation. vivo results, however, revealed no effect on the enzyme which suggests another mode of action. Epoxldatlon of cholesterol and membrane lysis may be alternative primary sites. If this were the case ecdysone metabolism of insects would probably also be effected. 

The morphological and functional development of the kidney has extensively been studied in rats. There is a rapid differentiation of the renal cortex after birth (83). The proximal segments of the tubules grow most rapidly, and micropuncture techniques have shown that glucose is reabsorbed solely by this segment of the tubule. Baxter and Yoffey have reported that in the newborn rat the peripheral region of the renal cortex is characterized by a neogenic zone of undifferentiated tissue, which cannot store the vital dye, trypan blue. By 28 days after birth, the tubules... 

Bucana CD, Giavazzi R, Nayar R, et al. Retention of vital dyes correlates inversely with the multidrug-resistant phenotype of adriamycin-selected murine fibrosarcoma variants. Exp Cell Res 1990 190(l) 69-75. 

Because the cells are viewed directly, this method enables a judgement to be made about the quality of the cell suspension, i.e. clumped or broken cells present. By diluting the cell suspension with a solution of a vital dye, e.g. trypan blue at a final concentration of 0.2 g/1 PBS-A (Appendix 1), and counting only the unstained cells a measure of the viable cell count is obtained. (N.B. Trypan blue is toxic and should not be allowed to come in contact with the skin.)... 

Typically, the integrity of the cell membrane is evaluated by identifying the percentage of cells taking up a vital dye termed trypan blue. Cells that take up trypan blue have defective cell membranes and therefore external loading or contact with materials that cause cells to have faulty membranes are likely to cause cell injury and death. Cell cytotoxicity is evaluated by contacting a surface of a material or the extract of a material or implant with cells in culture. If more than five percent of the cell population stains with dye after contact with the material or an extract then it is considered cytotoxic. 

Endpoint assays such as proliferation or cytotoxicity assays are routinely used for functional assessments. For these assessments, primary cells, transformed cells, or cells transfected with the target receptor are exposed to range of concentrations of the test article. Proliferation or cytoxicity is then measured using a variety of methods such as crystal violet vital dye staining, MTT/MTS incorporation, or a luminescence readout like ATP lite. In addition, assays that analyze phosphorylation of specific transcription factors, or release of specific cytokines and chemokines, are also common. Figure 9.5 illustrates the measure of functional consequences of receptor-test article interaction by quantifying cytokine release. Cells from the species under evaluation were cultured in the presence of serial dilutions of the test article or control reagents, and supernatants harvested for determination of cytokine levels by ELISA (i.e.,... 

In all these experimental models, the MTX effect could be divided in three phases (1) a rapidly developing Ca conductance through NSCC (2) uptake of vital dyes (ethidium, YO-PRO and POPO-3) and Fura-2 loss via a cytolitic-oncotic pore (COP) and (3) a secondaiy phase of vital dye uptake and Fura loss in which membrane permeability to larger molecules like LDH enzyme occurs. Fig. 4.4 shows permeability changes in a population study of bovine aortic endothehal cells (BAECs) treated with 0.3 nM MTX. Two phases of ethidium bromide uptake may be observed (steps 2 and 3 of the death process), the second phase correlating in time with LDH release (step 3 of death process). 

Widely used in the diagnosis of ocular surface disease, the understanding of the staining characteristics of rose bengal has evolved. Relatively recent evidence suggests that it is not a vital dye but one that may actually cause toxicity and cell death under certain circumstances. 

Medial obstruction Lateral tear overproduction Slit-lamp evaluation White light Vital dye staining... 

Kim J. The use of vital dyes in corneal disease. Curr Opin Ophthalmol 2000 11 241-247. 

Fig. 6. Binding of I-labeled protein A (PA) (0.1 ml 26,0(X) cpm added) to 10 line-10 (L-10) tumor cells sensitized with increasing amounts of rabbit antiserum to line-10 cells ( — ) or to the antigenically distinct line-1 (L-1) (O—O). These determinations of total bound IgG are compared to the cytotoxic (complement-fixing) activity of the antisera. Cytotoxicity was measured as uptake of the vital dye trypan blue following incubation of the antiline-10 (A—A) or anti-line-1 (A—A) sensitized line-10 cells with human complement. A relative concentration of 1 corresponds to a si dilution of antibody. From data given in Lan-gone et al. ...

The most common assay systems consist of cell cultures that are treated with the drug in the culture medium. Toxicity is commonly assessed by release of intracellular enzyme, for example, lactate dehydrogenase or aspartate transaminase, into the culture medium or by other indicators such as decreases in the rate of radiolabeled amino acid or nucteotide precursor incorporation into macromolecules. A decrease in the intracellular uptake of the vital dye, neutral red, has also... 

Continue incubation for 3 h to allow for incorporation of vital dye into surviving cells. 

Necrotic cells may be recognized morphologically by cell swelling and nuclear flocculation (Figure 4.4.2). Cultures with a high proportion of necrotic cells should have a relatively low number of cells with the ability to exclude vital dyes. 

Commonly used methodological variations to improve the sensitivity and accuracy of describing damage in these tests are inspection of the eyes with a slit lamp and instillation of the eyes with a vital dye (very commonly, fluorescein) as an indicator of increases in permeability of the corneal barrier. 

Vital dyes (trypan blue, eosin) fluorescence (Floechst, fluoresceins, rhodamine, ethidium bromide, propidium iodide) exogenous ( Cr release) endogenous (LDFI or alkaline phosphatase leakage, intracellular K+, lipid peroxidation)... 

As described by Reader S, Blackwell V, O Hara R, et al. (1989) A vital dye release method for assessing the short term cytotoxic effects of chemicals and formulations. Alternatives to Laboratory Animals 17 28-37. 

The EpiOcular model uses normal human-derived epidermal keratinocytes, which form a stratified squamous epithelium morphologically similar to that found in the cornea [36]. The main endpoint evaluated is cell viability measured by dehydrogenase conversion of the vital dye MTT (3-(4,5-Dimethyl-2-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Thiazolyl blue EINECS number 206-069-5, CAS number 298-93-1), into a blue formazan salt that is quantitatively measured after extraction from tissues. Two different protocols exist, the EpiOcular ET50 mainly developed for the testing of surfactants and surfactant-based mixtures, and the EpiOcular Eye Irritation Test (EIT) developed for a variety of chemistries. 

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