1. Composition of trypan blue working solution 1.1 Prepare the storage solution using 0.85% Nacl and 0.4% trypan blue. For example, use 100 mL 0.85% Nacl oxygen 400 mg trypan blue. Note: It is recommended to use pre-prepared serum-free cells or PBS immediate storage solution after dispensing 1.2 working solution to make a 0.04% trypan blue working solution. Note: Please alter the trypan blue working solution according to the actual situation. 2. Cell staining 2.1 Suspension cells: Collect cells by centrifugation, add PBS and wash twice, 5 minutes each time. Adherent cells: Discard the culture medium and add islet digested cells. After centrifugation and discarding the supernatant, add PBS and wash twice, 5 minutes each time. 2.2 Add 1 mL of trypan blue working solution and place it in the freezer for 5 minutes. 2.3 Centrifuge at 400 g for 3-4 minutes at 4°C and discard the supernatant. 2.4 Wash the cells twice with PBS, giving them five minutes each time. 2.5 After resuspending the cells with 1 mL of serum-free cells or PBS, the cell death rate can be more reliably assessed by counting directly under the cells or counting after taking photographs under the cells.

Absorption, Distribution and Excretion
In animal studies, trypanocyanin was observed to cross the walls of blood vessels in the iris and choroid, but not the walls of retinal vessels. Following subcutaneous or intraperitoneal injection, trypanocyanin was rapidly absorbed and widely distributed in mice and rats. Serum concentrations peaked within 2 hours; it appeared to bind to serum proteins and was rapidly excreted in the urine, while also being absorbed by the reticuloendothelial system. Trypanocyanin did not reach rat embryos but accumulated in the maternal reticuloendothelial system and placenta. Experiments using ring-labeled radioactive trypanocyanin showed no evidence of embryonic uptake of ¹⁴C. No teratogenic effects were observed after chorioallantoic placental formation, suggesting that yolk sac function plays an important role in pathogenesis. The dye was visible in visceral yolk sac cells. For more complete data on the absorption, distribution, and excretion of trypanocyanin (6 species), please visit the HSDB record page.

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Metabolisms/Metabolites
Trypan blue can be reduced in vitro by rat liver enzymes to o-toluidine and 2,8-diamino-1-naphthol-3,6-disulfonic acid.
Six azo dyes, including trypan blue, were reduced, N-acetylated, and N-conjugated. N,N'-diacetylated metabolites were not detected in rat urine.
Azo dyes based on β-diketone coupling components mainly exist in the form of tautomer hydrazones. A series of hydrazone dyes based on benzidine and its homologues were prepared…hydrazone dyes were resistant to enzymatic reduction in the mitochondrial supernatant (S-9) of hamster liver supplemented with FMN; under the same conditions, azo dyes (such as trypan blue) were rapidly reduced.
We performed metabolic experiments on rats, administering nine azo dyes based on dimethylbenzidine, dimethoxybenzidine, or dichlorobenzidine to determine whether free amine homologues, their monoacetyl or diacetyl metabolites, and alkaline hydrolysis conjugates were excreted in urine. Each dose was 2 mg, and urine samples were collected at intervals up to 96 hours. Peak concentrations of metabolites occurred at 0–12 hours or 12–24 hours post-administration; in seven of the nine cases, no metabolites were detected in urine after 48 hours. The lowest detectable concentration of all metabolites was 12 ppb or lower. In rats, all nine dyes were converted into measurable benzidine-based metabolites.
In vitro, the ability of phenobarbital-pretreated rat liver microsomes to reduce the azo groups of Amaranth, Sunset Yellow, Congo Red, Trypanosome Blue, Chloramine Sky Blue FF, and Direct Black 38 was determined. Amaranth and Sunset Yellow served as positive controls. Among dyes derived from benzidine (a carcinogen) or its homologues, only Direct Black 38 was significantly reduced; its reduction rate was approximately 10% of that of amaranth red. The mutagenicity of these dyes was tested in a Salmonella/microsomal assay, and only Direct Black 38 showed activity. The mutagenicity of this dye may be partly attributed to the mutagen 1,2,4-triaminobenzene. The mutagenic activity and azo reduction of Direct Black 38 were independent of the presence of oxygen. The mammalian liver likely plays only a minor or negligible role in the azo reduction of dyes derived from benzidine or its homologues.

Interactions
This study investigated the effects of L-glutamate on the lethal and teratogenic effects of trypan blue on rat embryos using Wistar albino rats. L-glutamate was added to the diet from day 2 to day 20 of gestation; from day 6 to day 10 of gestation, L-glutamate was suspended in sesame oil and administered via gavage. On day 8 of gestation, rats were given teratogenic doses of trypan blue either intraperitoneally (14 mg/kg maternal body weight) or subcutaneously (160 mg/kg). Following trypan blue injection, the daily intake of glutamate by rats ranged from 600 to 1500 mg per rat. Fetal examination was performed on day 20 of gestation. The results showed that glutamate failed to effectively protect rat embryos from the lethal and teratogenic effects of trypan blue. These results contradicted those observed in mice. Sesame oil alone caused embryonic death but not malformation.

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Non-human toxicity values
Oral LD50 in rats: 6200 mg/kg
Subcutaneous LD50 in mice: 267 mg/kg
Intravenous LD50 in mice: 328 mg/kg

[1]. Daly, M. L., DeRosa, C. A., Kerr, C., Morris, W. A., & Fraser, C. L. (2016). Blue thermally activated delayed fluorescence from a biphenyl difluoroboron β-diketonate. RSC Advances, 6(85), 81631–81635. doi:10.1039/c6ra18374c.

According to California labor law, commercial-grade trypan blue may be carcinogenic. Trypan blue is a blue-gray to dark blue powder. (NTP, 1992) Trypan blue is an organic sulfonate, the tetrasodium salt of 3,3'-[(3,3'-dimethylbiphenyl-4,4'-diyl)diazepine-2,1-diyl]bis(5-amino-4-hydroxynaphthalene-2,7-disulfonic acid). It is used as a histological dye, fluorescent dye, and carcinogen. It is an organic sulfonate and organic sodium salt. It contains trypan blue (4-). Trypan blue is an acidic azo dye commonly used as a staining agent to distinguish between living and dead cells. It turns dead cells blue while leaving living cells unstained. It is a known animal carcinogen and experimental teratogen. A diazonaphthalene sulfonate widely used as a staining agent. See also: Trypan blue (note moved to).

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Mechanism of Action
A dose of 50 mg trypan blue per kilogram of body weight appears to be the optimal teratogenic dose. A hallmark of trypan blue is that malformations are rare when treatment is administered after day 9 of gestation. This fact supports other evidence that its mechanism of action depends on the disruption of yolk sac nutrition. …Studies have confirmed that trypan blue may act on the nutritional function of the visceral yolk sac. The failure of trypan blue to act directly on the embryo is generally considered to be…
The absence of teratogenic effects after the formation of the chorioallantoic placenta also suggests that yolk sac function plays an important role in the pathogenesis. The dye can be visualized in visceral yolk sac cells. …The protein-trypan blue complex is concentrated in lysosomes. Trypan blue may interfere with normal embryonic nutritional processes by interfering with enzymatic digestion in yolk sac lysosomes.

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Therapeutic Uses
A diazonaphthalenesulfonate widely used as a staining agent.
A drug used to treat sleeping sickness. /Previous Uses/
0.15% MembraneBlue is indicated for use in ophthalmic surgery as an adjunct to staining the epiretinal membrane during vitrectomy to facilitate tissue removal.
/Experimental Treatment/ Trypan blue can be used to stain the superior oblique tendon to facilitate identification and delineation of its insertion point, thereby reducing surgical difficulty.
For more complete data on the therapeutic uses of trypan blue (6 types), please visit the HSDB record page.
Drug Warnings
Adverse reactions reported after use of VisionBlue include discoloration of high-water-content intraocular lenses (see Contraindications) and accidental staining of the posterior capsule and vitreous surface. Posterior capsule staining or vitreous surface staining is usually self-limiting and can last up to one week.
It is currently unknown whether this drug is excreted into human breast milk. Because many drugs are excreted into human breast milk, caution should be exercised when using trypan blue in breastfeeding women.
VisionBlue is contraindicated if a dehydrated (dry) hydrophilic acrylic intraocular lens is planned for implantation, as the dye may be absorbed by the lens and stain it.
It is recommended to thoroughly flush the anterior chamber immediately after injection to remove any excess VisionBlue from the eye.
FDA Pregnancy Risk Classification: C/Risk cannot be ruled out. There is a lack of adequate, well-controlled clinical studies, and animal studies have not shown any risk to the fetus or lack relevant data. There is a possibility of fetal harm if this medication is used during pregnancy. However, the potential benefits may outweigh the potential risks.

C34H24N6NA4O14S4

960.8052

959.982

72-57-1

6296

Light brown to black solid powder

1.007 g/mL at 20 °C

>300 °C(lit.)

10.785

4

20

5

62

1790

0

GLNADSQYFUSGOU-UHFFFAOYSA-J

InChI=1S/C34H28N6O14S4.4Na/c1-15-7-17(3-5-25(15)37-39-31-27(57(49,50)51)11-19-9-21(55(43,44)45)13-23(35)29(19)33(31)41)18-4-6-26(16(2)8-18)38-40-32-28(58(52,53)54)12-20-10-22(56(46,47)48)14-24(36)30(20)34(32)42;;;;/h3-14,41-42H,35-36H2,1-2H3,(H,43,44,45)(H,46,47,48)(H,49,50,51)(H,52,53,54);;;;/q;4*+1/p-4

tetrasodium;5-amino-3-[[4-[4-[(8-amino-1-hydroxy-3,6-disulfonatonaphthalen-2-yl)diazenyl]-3-methylphenyl]-2-methylphenyl]diazenyl]-4-hydroxynaphthalene-2,7-disulfonate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~25 mg/mL (~26.02 mM)
H2O : ~1 mg/mL (~1.04 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)