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
Trypan blue ... was observed in animals to pass from bloodstream through walls of vessels of iris & choroid, but not through walls of retinal vessels.
Following sc or ip injections into mice or rats, trypan blue is rapidly absorbed & widely distributed throughout the body. Max serum concentrations are ... within 2 hr; it appears bound to serum proteins with ... rapid excretion in urine & uptake by the reticulo-endothelial system.
Trypan blue never reaches the rat embryo, but accumulates in maternal reticuloendothelial system and in the placenta.
Experiments with ring-labelled radioactive trypan blue did not give evidence of any embryonic incorporation of the (14)C. The absence of teratogenic action after the initiation of chorio-allantoic placentation also indicated that yolk sac function was important in pathogenesis. The dye can be visualized in the cells of the visceral yolk sac.
For more Absorption, Distribution and Excretion (Complete) data for Trypan blue (6 total), please visit the HSDB record page.

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Metabolism / Metabolites
/Trypan blue/ is reduced in vitro by a rat liver enzyme to ortho-tolidine & 2,8-diamino-1-naphthol-3,6-disulfonic acid.
Six azo dyes, including trypan blue, were reduced, N-acetylated, and N-conjugated. No N,N'-diacetylated metabolites were detected in rat urine.
Azo dyes based on beta-diketone coupling components exist preferentially as the tautomeric hydrazones. A series of hydrazone dyes based on benzidine and benzidine congeners ... was prepared. The hydrazone dyes were resistant to enzymatic reduction by FMN supplemented hamster liver post-mitochondrial supernatant (S-9); under identical conditions, azo dyes such as trypan blue were rapidly reduced.
Metabolism experiments were conducted with rats dosed with 9 azo dyes based on dimethyl-, dimethoxy-, or dichlorobenzidine to determine whether the free amine congeners, their monoacetyl or diacetyl metabolites, or alkaline hydrolyzable conjugates were excreted in the urine. 2-mg doses were administered and urine samples were collected at intervals up to 96 hr. Peak levels of metabolites were excreted either 0-12 or 12-24 hr after administration and, in 7 of 9 instances, no metabolites persisted in the urine after 48 hr. Minimum detectable levels of all metabolites were 12 ppb or less. All 9 dyes were converted to measurable levels of their benzidine-congener-based metabolites in rats.
The ability of rat liver microsomes from phenobarbitone pretreated animals to reduce the azo groups of amaranth, sunset yellow, congo red, trypan blue, chloramine sky blue FF and direct black 38 was measured in vitro. The dyes amaranth and sunset yellow acted as positive controls. Of the dyes derived from (the carcinogen) benzidine or its congeners, only direct black 38 was reduced to an appreciable extent; the rate of reduction was 10% of that for amaranth. The dyes were tested for mutagenicity in the Salmonella/microsome assay, the only active compound being direct black 38. Mutagenicity of this dye may be due in part to the mutagen 1,2,4-triaminobenzene. Mutagenic activity and azo-reduction of direct black 38 was independent of the presence of oxygen. Mammalian liver may play only a minor or negligible role in the azo-reduction of dyes derived from benzidine or its congeners.

Interactions
The effect of L-glutamic acid on the embryolethal and teratogenic action of trypan blue was investigated in Wistar albino rats. L-glutamic acid was either incorporated into the diet, from gestation day 2 to day 20, or suspended in sesame oil and administered by gavage, from gestation day 6 to day 10. A teratogenic dose of trypan blue was injected at day 8 of pregnancy, either intraperitoneally (14 mg/kg maternal body weight) or subcutaneously (160 mg/kg). The amount of glutamic acid consumed, after the injection of trypan blue, ranged from 600 to 1,500 mg/rat/day. At day 20, the fetuses were examined. Glutamic acid failed consistently to protect the rat embryo against the lethal and teratogenic action of trypan blue. These results are in contrast to those obtained in mice. The administration of sesame oil alone was found to cause embryonic death but not malformations.

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Non-Human Toxicity Values
LD50 Rat oral 6200 mg/kg
LD50 Mouse sc 267 mg/kg
LD50 Mouse iv 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.

Trypan Blue (Commercial Grade) can cause cancer according to California Labor Code.
Trypan blue is a bluish-gray to dark blue powder. (NTP, 1992)
Trypan blue is an organosulfonate salt that is the tetrasodium salt of 3,3'-[(3,3'-dimethylbiphenyl-4,4'-diyl)didiazene-2,1-diyl]bis(5-amino-4-hydroxynaphthalene-2,7-disulfonic acid). It has a role as a histological dye, a fluorochrome and a carcinogenic agent. It is an organosulfonate salt and an organic sodium salt. It contains a trypan blue(4-).
Trypan Blue is an acid azo dye commonly used as a stain to distinguish viable from non-viable cells. It turns dead cells blue and viable cells unstained. It is a known animal carcinogen and an experimental teratogen.
A diazo-naphthalene sulfonate that is widely used as a stain.
See also: Trypan Blue (annotation moved to).

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Mechanism of Action
A dose of 50 mg per kg /of trypan blue/ appears to be the optimum teratogenic dose. A characteristic observation with trypan blue is that with treatment after the 9th day of gestation defects are rare. This fact has supported other evidence that the mechanism of action was dependent on disruption of yolk sac nutrition. ... Studies have produced evidence indicating a possible action of trypan blue on a nutritive function of the visceral yolk sac. The failure of the trypan blue to act directly upon the embryo is generally held ... .
The absence of teratogenic action after the initiation of chorio-allantoic placentation also indicated that yolk sac function was important in pathogenesis. The dye can be visualized in the cells of the visceral yolk sac. ... . The protein-trypan blue complex is concentrated in lysosomes. Through disruption of the enzymatic digestive process in the yolk sac lysosome, trypan blue may interfere with normal embryonic nutritive processes.

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Therapeutic Uses
A diazo-naphthalene sulfonate that is widely used as a stain.
A therapeutic agent in the treatment of sleeping sickness. /Former use/
MembraneBlue 0.15% is indicated for use as an aid in ophthalmic surgery by staining the epiretinal membranes during ophthalmic surgical vitrectomy procedures, facilitating removal of the tissue.
/Experimental Therapy/ Trypan blue can be used to stain the superior oblique tendon for easy identification and delineation of it at its insertion, making the current surgical technique less difficult.
For more Therapeutic Uses (Complete) data for Trypan blue (6 total), please visit the HSDB record page.

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Drug Warnings
Adverse reactions reported following use of VisionBlue include discoloration of high water content hydrogen intraocular lenses (see Contraindications) and inadvertent staining of the posterior lens capsule and vitreous face. Staining of the posterior lens capsule or staining of the vitreous face is generally self limited, lasting up to one week.
It is not known whether this drug is excreted in human milk. Because many drugs are excreted in human milk, caution should be exercised when trypan blue is administered to a nursing woman.
VisionBlue is contraindicated when a non-hydrated (dry state), hydrophilic acrylic intraocular lens is planned to be inserted into the eye because the dye may be absorbed by the intraocular lens and stain theintraocular lens.
It is recommended that after injection all excess VisionBlue be immediately removed from the eye by thorough irrigation of the anterior chamber.
FDA Pregnancy Risk Category: C /RISK CANNOT BE RULED OUT. Adequate, well controlled human studies are lacking, and animal studies have shown risk to the fetus or are lacking as well. There is a chance of fetal harm if the drug is given during pregnancy; but the potential benefits may outweigh the potential risk./

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.)