Trypan Blue

TB is an anionic hydrophilic azo dye which has the molecular formula C34H24N6Na4O14S4 and a molecular weight of 960 Da. TB crosses the cell membranes of dead cells only, thereby staining dead tissues/cells blue.

From: Retinal Pharmacotherapy , 2010

Macular Hole

Andrew P. Schachat MD , in Ryan's Retina , 2018

Trypan Blue.

Trypan blue (TB) stains well the ERM but poorly the ILM. Nevertheless, it remains an alternative to ICG. To improve TB staining of the ILM, it must be used after fluid–gas exchange. Alternatively, it is easier to mix the dye isovolumetrically in 10% glucose to create heavy TB, which falls onto the posterior pole and results in an acceptable staining after a 2-min contact. 153 However, it has been suggested that this may also increase its osmolarity to toxic levels. 154,155 Alternatively, ready-to-use solutions combining TB with a viscosity- and density-increasing agent or another dye are also available in some countries. In most studies, TB showed no signs of toxicity to the RPE or neuronal tissue. 156

Nanomedicine

I-Ju Fang , Brian G. Trewyn , in Methods in Enzymology, 2012

2.6.3 The purpose of the trypan blue

Trypan blue is a stain used to quantify live cells by labeling dead cells exclusively. Because live cells have an intact cell membrane, trypan blue cannot penetrate the cell membrane of live cells and enter the cytoplasm. In a dead cell, trypan blue passes through the porous cell membrane and enters the cytoplasm. Under light microscopy analysis, only dead cells have a blue color. Trypan blue is also used to eliminate false positives from occurring during cell counting by flow cytometry ( Radu et al., 2004; Slowing et al., 2006; Trewyn et al., 2008). Fluorescein labeled MSN can be either internalized by cells or they can physically adsorb to the exterior of the cell membrane. When these two events occur, it is often difficult to distinguish between them by flow cytometry. By adding trypan blue to the solution, the extracellular FITC-MSN material is quenched, excluding them from the quantification. Only live cells that have internalized the FITC-MSN report a positive result. Trypan blue cannot penetrate the membrane of live cells, it will quench all the fluorescence from the physically adsorbed MSN on cell membrane. Thus, when Trypan blue is utilized, the fluorescence detected by flow cytometry is from the cells with internalized fluorescent MSN or cell autofluorescence. The concentration of the trypan blue solution used is typically 0.4% (w/w).

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The use of vital dyes during vitreoretinal surgery – chromovitrectomy

Michel Eid Farah MD , ... Eduardo Büchele Rodrigues MD , in Retinal Pharmacotherapy, 2010

TRYPAN BLUE

TB has been proposed to stain preretinal tissues such as ILM and ERM in chromovitrectomy (Figure 48.2). Although the use of TB for ILM staining may also be feasible, in our experience ILM visualization is much more difficult than with the ICG- or BriB-guided procedure. Currently, state-of-the-art TB usage recommends blue dye application mainly for ERM staining. TB exhibits outstanding affinity for ERM because of the strong presence of dead glial cells within those membranes. TB staining of the ERM may minimize mechanical trauma to the retina during ERM removal and should allow the recognition of the whole extent of the ERM, which has led to good surgical outcomes and minimization of the recurrence of ERM in several clinical studies. 11

The usefulness of intracameral or intravitreal injection of TB to highlight vitreous gel has been recently proposed. The blue dye in various doses may enhance the ability to detect both the prolapsed vitreous to the anterior chamber and the posterior vitreous remaining in the vitreous cavity. However, in one comparative analysis TB demonstrated an inferior ability to stain the vitreous in comparison to TA and FS. 12

TB in vitreoretinal surgery was initially proposed for injection after an air–fluid exchange, allowing for better dye dropping on to the retinal surface. To enhance dye penetration on to retinal surface in an air–fluid exchange, TB may be mixed with glucose at higher than 5%, for instance 10% or 25%, thereby creating a heavy TB, denser than water. However, high glucose concentrations should be avoided; for instance, glucose 50% has a highly toxic osmolarity of 2020 mOsm/l. Experimental injection of 0.05 ml of a 1000 mOsm solution caused rapid whitening of the posterior retina followed by the development of a large detachment and permanent retinal degeneration. Indeed, osmolarity should be taken into consideration in planning the amount and location of any vitreous injection of dyes and drugs.

Consecutive clinical studies have revealed that TB exerts little or no toxic effect on the retina. For ERM surgery, TB caused no RPE defects or signs of retinal toxicity in most studies in the literature until now. Histopathological analysis of excised ERM showed no retinal cells on the retina side of the ERM or signs of apoptosis, while functional analysis by multifocal ERG also showed no signs of retinal toxicity. Comparative studies evaluated the anatomical and visual outcomes after vitrectomy and ILM peeling for treatment of patients with macular hole using ICG or TB. Their success rate for MH closure was the same; however, visual recovery has been better in the TB group.

In animal experiments, TB has demonstrated a reasonably good retinal biocompatibility in most investigations at the concentration proposed for intravitreal application from 0.06% to 0.15%. Grisanti et al. used fresh hemisected porcine eyes and applied TB 0.15% to the posterior pole after vitreous removal followed by illumination with a standard surgical light probe and source at maximum power for 10 minutes; the procedure caused no histologically detectable damage. These results imply that TB at concentrations of 0.15% or lower represents a safe adjuvant in vitreoretinal surgery. 13

Various laboratory studies have evaluated the retina biocompatibility of TB alone or in comparison to ICG. In cultured human RPE and Müller cells, some authors have reported that exposure to TB at concentrations up to 0.3% in vitro induced no toxic effect. 14 In accordance with these studies, recent investigations revealed that TB is toxic to cultured RPE cells at concentrations higher than 0.5%. However, ICG may cause more toxicity to the retina, especially to human RPE cell cultures, than TB, independent of any phototoxic potentiating effect of fiberoptic light or solvent toxicity. Our research group showed that subretinal injection of 0.05% ICG results in more substantial retinal damage than that associated with subretinal injection of 0.15% TB. 15

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Antiparasitic drugs

Stephen W Page , in Small Animal Clinical Pharmacology (Second Edition), 2008

Trypan blue

3,3′-[(3,3′-dimethyl(1,1′-biphenyl)-4,4′-diyl)bis(azo)]bis(5-amino-4-hydroxy-2,7-naphthalenedisulfonic acid) tetrasodium salt.

Trypan blue is an antiprotozoal drug first used to treat Babesia infection in 1909 and still commonly used to treat Babesia canis. The complex chemical structure has been progressively simplified, yielding such other widely used drugs as imidocarb. Trypan blue is administered IV at a rate of 10 mg/kg as a 1% solution. Babesia are cleared from the blood within 24–48 h, corresponding to noticeable signs of recovery in dogs with uncomplicated cases. Trypan blue can cause blue discoloration of mucous membranes and plasma following administration and there is a potential for relapse of babesiosis.

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DEPLETION OF MONONUCLEAR PHAGOCYTES: PITFALLS IN THE USE OF CARBONYL IRON, CARRAGEENAN, SILICA, TRYPAN BLUE, OR ANTIMONONUCLEAR PHAGOCYTE SERUM

Paul A. LeBlanc , Stephen W. Russell , in Methods for Studying Mononuclear Phagocytes, 1981

A Introduction

Trypan blue has been reported to interfere with the rejection of allogeneic skin ( 18) and tumors (19), as well as the elimination of antigenic, syngeneic neoplasms (20). Treatment of animals with the dye has also resulted in reduced resistance to Trypanosoma musculi (21). Because of these, and other findings, it has been suggested that trypan blue may be a selective inhibitor of macrophage function (18). While there is little doubt that this agent affects macrophages, as will be seen from the comments in Section V.D, it has a wide spectrum of effects and should not, therefore, be viewed as selectively affecting macrophages.

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Models and Methods for In Vitro Toxicity

Abhishek K. Jain , ... Alok K. Pandey , in In Vitro Toxicology, 2018

Trypan Blue Exclusion Assay

Trypan blue exclusion assay was one of the most common and earliest method used for cell viability measurement [40]. It is impermeable for the normal cell membrane and therefore only enters the cell with compromised membrane. After entering the cell, it binds into the intracellular proteins and renders them bluish color. It also helps in the identification and counting of live or dead cells in the given cell population. The viable cells are shown small, rounded, and refractive, whereas dead cells are shown swollen, large, and dark blue.

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T

Michel Millodot OD, PhD, DOSc(Hon), FAAO, FCOptom , in Dictionary of Optometry and Vision Science (Eighth Edition), 2018

Tr

trypan blue

A blue dye used in cataract surgery; it is applied directly onto the anterior lens capsule, which it stains, thereby aiding its visualization and facilitating its removal. It is also used in eye banks to evaluate the quality of the endothelium of human donor corneas as it stains the nuclei of damaged or dead cells.

See phacoemulsification .

Tscherning ellipse

See ellipse, Tscherning .

t-test

See Student's t-test .

tubercle, lacrimal

See lacrimal tubercle .

tubercle, lateral orbital

A small elevation on the orbital surface of the zygomatic bone just behind and within the orbital margin, about 11 mm below the suture of the zygomatic and frontal bones. It serves as an attachment for the check ligament of the lateral rectus muscle, the lateral palpebral ligament, the suspensory ligament of Lockwood and the levator palpebrae superioris muscle. Syn. Whitnall's tubercle.

See ligament of Lockwood ; ligament, palpebral .

tubercle, Whitnall's

See tubercle, lateral orbital .

tuberculosis

Chronic infection caused by the microorganism Mycobacterium tuberculosis, which causes primarily a pulmonary disease but also of various ocular tissues.

See disease, Eales' ; keratitis, interstitial ; neuropathy, optic ; uveitis, bacterial .

tuberous sclerosis

See sclerosis, tuberous .

tuck procedure

See procedure, tuck .

tumbling E chart

See chart, E .

tungsten-halogen lamp

See lamp, halogen .

tunica vasculosa lentis

See artery, hyaloid .

tunnel vision

See vision, tunnel .

Turcot's syndrome

See syndrome, Turcot's .

turgescence

The swelling of a tissue, usually as a result of water accumulation.

See deturgescence .

Turk's disease

See syndrome, Duane's .

Turner's syndrome

See syndrome, Turner's .

Turville infinity balance test

See test, Turville infinity balance .

twitch, eyelid

See myokymia .

twilight vision

See vision, mesopic .

two visual systems theory

See theory, two visual systems .

Tyndall effect

See effect, Tyndall .

typoscope

A reading shield made of black material in which there is a rectangular aperture allowing one or more lines of print to be seen. It reduces extraneous light reflected from the surface of the paper and assists in staying on the correct line (Fig. T24). It can be helpful for people with low vision who have, for example, media involvement. Recent models embody built-in lighting to provide even and controlled illumination. Syn. reading slit.

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Cell preparation for 3D bioprinting

A. Al-Sabah , ... C. Thornton , in 3D Bioprinting for Reconstructive Surgery, 2018

4.2.3 Viability

a.

Trypan blue exclusion method

Whether cells are expanded for experimental processes such as testing a new scaffold or prepared to be implanted in patients, viability tests are necessary to determine that the growth conditions or prolonged culture of cells is not resulting in cell death [25]. There are multiple viability tests available to determine cell viability. Trypan blue exclusion method is one of the earliest and simplest viability assays. Trypan blue is a negatively charged dye which only stains cells with a compromised cell membrane, hence indicating cell death [26]. In contrast, viable cells are absent of trypan blue due to both the cell membrane and dye being negatively charged.

b.

MTT assay

In addition to dye exclusion, some viability assays use metabolic activity as an indicator of cell viability. One of these assays is the MTT assay which is a commonly used colorimetric viability assay based on detecting cellular metabolic activity. The assay utilizes 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT), a yellow tetrazolium salt, to evaluate the efficiency of mitochondrial enzymes. MTT is reduced via mitochondrial dehydrogenases to an insoluble purple formazan product [27]. As the formazan product is insoluble it is entrapped within live cells. The amount of formazan produced would be proportional to the number of viable cells. A spectrophotometer is used to measure the absorbance values of formazan to determine cell number. Similar in principle to the MTT assay, Alamar Blue assesses cell growth and viability via cell metabolism [28,29]. Alamar Blue is a nontoxic permeable dye which is reduced by cells to a pink fluorescence product which is then measured by a spectrophotometer. The nontoxic nature of Alamar Blue allows the monitoring of cells in culture over time, hence providing information regarding proliferation rates as well as viability [30].

c.

Live/dead assay

The live/dead assay is the most informative viability assay available which presents information regarding the number of viable and nonviable cells, cell morphology, and the distribution of cells. The live/dead assay is a dual color fluorescence assay which discriminates viable from dead cells based on intracellular esterase activity [31]. The two dyes used in this assay are calcein acetoxymethyl ester (AM) and ethidium homodimer. Viable cells have an intact cell membrane where calcein AM, a nonfluorescent dye, is able to permeabilize and be hydrolyzed by intracellular esterases into green fluorescence. In contrast, ethidium homodimer, a nonpermeable red fluorescent dye, detects nonviable cells by penetrating the damaged cell membrane and binding to DNA [32].

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EFFECTS OF ISOLATION AND PURIFICATION PROCEDURES ON THE VIABILITY AND PROPERTIES OF TESTIS LEYDIG CELLS

B.A. COOKE , ... C.J. Dix , in Hormonal Steroids: Proceedings of the Sixth International Congress on Hormonal Steroids, 1983

2 Viability of the isolated cells

The exclusion of trypan blue is extensively used as a parameter for determining the viability of isolated cells. However, cellular exclusion of this large molecule does not necessarily indicate that the cells are viable. Tennant[ 19] found that trypan blue exclusion assays gave grossly inaccurate and misleading results following traumatic treatment of epithelial cells and tumour lymphocytes (e.g. dispersal of cell monolayer by trypsinization and EGTA treatment) and suggested that the cell membrane may remain "intact", although the cells were no longer capable of growth or multiplication. We have therefore investigated an alternative method of measuring cell viability by determining the uptake of pyridine nucleotides. We have compared the permeability of frozen and unfrozen cells to NAD+ and NADH using histochemical reactions for 3β-hydroxysteroid dehydrogenase and diaphorase [20].

Crude preparations of rat testis interstitial cells were separated into the 3 bands (I, II and III) on 0–90% Percoll gradients. In the crude preparation and in bands I and II 35–70%, and in band III 13%, of fresh cells were diaphorase positive (Fig. 2). However, less than 10% of any of these cell preparations took up trypan blue. In all fractions 90% of the frozen cells were diaphorase positive. The histochemical reaction was positive for less than 1% of the crude cells in the absence of NADH.

Fig. 2. Viability of rat testes cells after separation on Percoll gradients. Crude preparations of rat testis interstitial cells were separated into 3 bands on 0–90% Percoll gradients. The percentages of 3β-hydroxysteroid dehydrogenase and diaphorase positive cells were estimated by histochemistry before and after freezing of the cell suspensions. The results given are from one of 2 experiments which both gave similar results. Each value is the mean ± SD, n = 5 where n is the number of fields counted

 (from Aldred and Cooke[20]).

To assess the effect of BSA on the trypan blue test, the proportion of cells staining blue in the absence and presence of BSA (0.1% w/v) was determined. With BSA present 4.4 ±.3.0% of the crude cells were positively stained, compared with 1.9 ± 1.3% of crude cells which had been washed free of BSA (means ± SD, n = 5): approx. 50% of these cells were diaphorase positive. This indicates that the presence of 0.1% BSA in the media is not responsible for the difference in the proportion of "damaged" cells when determined by trypan blue exclusion and NADH exclusion. When the cells were incubated for 30 min at 37°C 4–5% of the crude cells stained blue compared with approx. 2% after 5 min incubation at room temperature.

Approximately 10% of the fresh cells in all fractions were 3β-hydroxysteroid dehydrogenase positive compared with 15–30% of the frozen cells in the unfractionated cells and in bands I and II, and 90% of the frozen cells in band III (Fig. 2). The histochemical reaction was positive for less than 1% of the crude cells in the absence of 5α-androstan 3β-ol-17-one. Assuming that cells staining for 3β-hydroxysteroid dehydrogenase were Leydig cells, the crude preparation contained 15, band I 7 and band II 4 million Leydig cells of which approx. 50% were viable. Band III contained approx. 3 × 106 viable Leydig cells (Fig. 3). Most of the nucleated cells centrifuged were recovered; the larger fraction appearing in band I with smaller amounts in bands II and III (Fig. 3).

Fig. 3. Recovery of nucleated and Leydig cells from Percoll gradients. Crude preparations of rat testis interstitial cells were separated into 3 bands on 0–90% Percoll gradients and the number of nucleated cells in each band determined. Cells staining for 3β-hydroxysteroid dehydrogenase were classified as Leydig cells, cells which stained prior to freezing were classified as "non-viable" Leydig cells and cells which stained only after freezing were classified as "viable" Leydig cells. The results given are from one of 2 experiments which both gave similar results. Each value is the mean ± SD, n = 5, where n is the number of fields counted

 (from Aldred and Cooke[20]).

To assess the effect of cell damage on the distribution of Leydig cells on Percoll gradients, band III cells were isolated from a gradient, washed and frozen with an equal volume of 6% dextran for 48 h. After thawing at 20°C and washing to remove the dextran, the cell suspension was applied to a second gradient and centrifuged. The cells isolated were compared with those obtained by purification of unfrozen band III cells on a second gradient. In one of two experiments giving similar results it was found that 95.4 ± 1.7% of the frozen cells compared with 8.0 ± 2.9% of the fresh cells were damaged when assessed by diaphorase histochemistry. The 3β-hydroxysteroid dehydrogenase histochemical reaction was positive for 85.0 ±4.1% of the frozen cells and 4.2 ± 1.5% of the fresh cells (means ± SD, n = 5 fields counted). After centrifugation on a second Percoll gradient, the frozen cells formed a distinct band (1.037 g/ml) above the band containing the fresh cells (1.076 g/ml). 95% of the total frozen and 87% of the total fresh nucleated cells applied to the second gradients were recovered. The lower density cells did not produce testosterone after stimulation with LH: the higher density cells produced 251.3 ± 9.9ng/106 nucleated cells/2 h (mean ± SD, n = 3 determinations).

Although pyridine nucleotides and nitroblue tetrazolium rapidly enter cells that have been rendered permeable by freezing, the intact cell membrane and mitochondrial membrane are impermeable to these large molecules [21, 22]. It can therefore be concluded that cells which stain for diaphorase or 3β-hydroxysteroid dehydrogenase prior to freezing have incurred membrane damage during the preparation and purification procedures inspite of the fact that they exclude trypan blue. We found that cell viability determined by trypan blue exclusion was always less than 10% of the cells in any fraction compared with 10–70% of the cells, depending on the cell fraction, when determined by NADH exclusion.

After purification on Percoll gradients most of the damaged cells were present in bands I and II. Band III contained mainly viable Leydig cells. It is apparent therefore that, although viable Leydig cells are also present in bands I and II, the Leydig cell heterogeneity previously reported [1, 5–8] may in part be due to cell damage. Pertoft et al. [23] have reported that cell damage results in a decrease in cell density, therefore damaged Leydig cells will appear in bands I and II on the Percoll gradient. This is confirmed by the demonstration that band III cells that were damaged by freezing were less dense when separated on Percoll gradients than undamaged cells. This change in density was accompanied by an inability to produce testosterone after stimulation with LH. These data are consistent with the studies reported above (section 1) which showed that mechanical dispersal of rat testes prior to collagenase treatment decreases the steroidogenic capacity and adenylate cyclase activity of the purified Leydig cell but has little or no effect on LH receptors [9].

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Nitric Oxide, Part G Oxidative and Nitrosative Stress in Redox Regulation of Cell Signaling

Orazio Cantoni , ... Liana Cerioni , in Methods in Enzymology, 2008

2.2 Cytotoxicity assay

Cytotoxicity is determined with the trypan blue exclusion assay immediately after treatments. Briefly, an aliquot (50 μl) of the cell suspension is diluted 1:1 (v/v) with 0.4% trypan blue and viable cells are counted with a hemocytometer. Note that the toxic treatments employed in this study reduce the number of viable cells without a parallel increase in the number of trypan blue positive cells, as cell lysis is very fast. Indeed, these cells remain viable for at least 30 to 40   min and then undergo an about a 3- to -5-min process in which they first swell and then rapidly lose their membrane integrity and lyse.

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