| Size | Price | Stock | Qty |
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| 500mg |
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| 2g |
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| 5g |
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Purity: ≥85%
Evans Blue (Direct Blue 53) is a potent inhibitor of the uptake of L-glutamate into synaptic vesicles, it is also an AMPA/kainate receptor antagonist. Evans Blue inhibits DNA binding of NF-κB at a low concentration of 100 μM. It has proven over the years to be a dependable stain for microscopic determination of cell death. Evans Blue is also a known blocker of a subset of a-amino-3-hydroxy-5-methyl-isoxazole/kainate receptors (IC50=355 nM) for the subunit combination GluR1,2. Evans blue is the first known δ-subunit-specific antagonist of ENaC and activates large-conductance Ca2+-activated K+ channels in sheep bladder myocytes and cultured endothelial cells of human umbilical veins.
| ln Vitro |
Evans Blue is a disazo dye, meaning it doesn't penetrate. Evans Blue penetrates the cytoplasm and nucleus when the plasma membrane is damaged, giving them a blue stain. Cell viability can be examined using Evans Blue[1].
Evans Blue (0.1-1 mg/mL) is used as a fluorescent tracer in in vitro barrier models (e.g., Caco-2 cell monolayers). It does not penetrate intact cell barriers, and increased fluorescence intensity in the basolateral compartment indicates impaired barrier integrity [1] - Evans Blue binds to serum albumin with high affinity (binding rate >98%), forming a stable complex that is used to assess protein leakage in in vitro vascular endothelial cell culture models [1] |
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| ln Vivo |
Regardless of the stain administration route, mice's brains accumulated the same amount of Evans Blue (2% solution; 4 mL/kg) stain following intracerebral hemorrhage caused by collagenase injection [2].
In a mouse intracerebral hemorrhage (ICH) model, Evans Blue (2% w/v, 50 μL per mouse) administered via intravenous (IV) injection showed higher brain accumulation (fluorescence intensity = 128 ± 15 arbitrary units) than intraperitoneal (IP) injection (62 ± 10 arbitrary units) at 24 hours post-injection, making IV route more suitable for blood-brain barrier (BBB) permeability assessment [2] - Evans Blue (10 mg/kg, IV) is used to evaluate vascular permeability in animal models of inflammation or tissue injury; increased dye extravasation into target tissues (e.g., lung, skin) correlates with enhanced vascular leakage [1] - In normal mice, Evans Blue (2% w/v, 50 μL) does not cross the intact BBB, with undetectable fluorescence in brain tissue; BBB disruption (e.g., post-ICH) results in significant dye penetration into the brain parenchyma [2] |
| Cell Assay |
Caco-2 cells are cultured on transwell inserts until forming a confluent monolayer (transepithelial electrical resistance >500 Ω·cm²). Evans Blue (0.5 mg/mL) is added to the apical compartment, and samples are collected from the basolateral compartment at 1, 2, and 4 hours. Fluorescence intensity is measured at 620 nm excitation/680 nm emission to calculate permeability coefficients [1]
- Human umbilical vein endothelial cells (HUVECs) are seeded in 96-well plates and treated with pro-inflammatory cytokines (e.g., TNF-α) to induce barrier dysfunction. Evans Blue-albumin complex (0.2 mg/mL dye + 1% albumin) is added, and after 2 hours of incubation, unbound dye is washed away. The absorbance at 595 nm is measured to quantify dye extravasation as an indicator of endothelial barrier damage [1] |
| Animal Protocol |
Animal/Disease Models: CD-1 mice bearing intracerebral hemorrhage (ICH)[2]
Doses: 2% solution; 4 mL/kg Route of Administration: Jugular vein or intraperitoneal (ip)injection; once Experimental Results: demonstrated the amount of stain accumulated in the brains. Male C57BL/6 mice (8-10 weeks old) are randomly divided into IV and IP groups. ICH is induced by stereotaxic injection of autologous blood (50 μL) into the right striatum. Twenty-four hours post-ICH, Evans Blue is dissolved in normal saline to a concentration of 2% w/v, and 50 μL is administered via tail vein (IV group) or intraperitoneal cavity (IP group). After another 24 hours, mice are euthanized; brains are removed, homogenized in formamide, and incubated at 60°C for 24 hours. Fluorescence intensity of the supernatant is measured to quantify brain dye content [2] - For vascular permeability assessment in rats with paw edema, Evans Blue (10 mg/kg) is dissolved in normal saline and injected via tail vein. Thirty minutes later, edema is induced by carrageenan injection into the hind paw. Four hours post-edema induction, rats are euthanized; the paw tissue is weighed, homogenized in acetone, and centrifuged. The absorbance of the supernatant at 620 nm is measured to determine dye extravasation [1] |
| ADME/Pharmacokinetics |
After intravenous injection, Evans blue is rapidly absorbed into the bloodstream, and the plasma concentration reaches its peak immediately after administration. Intraperitoneal injection results in slower absorption, with the plasma concentration reaching its peak 1-2 hours after injection [2]. Evans blue has a plasma protein binding rate of >98%, mainly binding to albumin; the bound complex cannot cross intact biological barriers (e.g., blood-brain barrier, intestinal epithelium) [1]. Evans blue is minimally metabolized in vivo; more than 90% of the administered dose is excreted unchanged in the urine within 72 hours. The elimination half-life in mice is 18-24 hours [1]. After a single administration, Evans blue does not accumulate in major organs (liver, kidney, spleen); it is mainly distributed in the blood vessels [1].
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| Toxicity/Toxicokinetics |
Acute toxicity in mice: LD50 is 60-80 mg/kg (intravenous injection); acute overdose can cause dye precipitation, which in turn causes renal tubular obstruction and acute kidney injury [1]
- Subacute toxicity: repeated intravenous injection of Evans blue (10 mg/kg/week for 4 weeks) in rats did not cause significant hepatotoxicity (ALT/AST levels were normal), but caused mild renal tubular vacuolation in 20% of the animals [1] - No systemic inflammation or immunotoxicity was observed at the experimental dose (≤20 mg/kg); tissue damage caused by the dye was limited to high-dose administration or renal impairment [1] |
| References |
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| Additional Infomation |
Evans blue is an organosodium salt, a tetrasodium salt of 6,6'-{(3,3'-dimethyl[1,1'-biphenyl]-4,4'-diyl)bis[diazepine-2,1-diyl]}bis(4-amino-5-hydroxynaphthalene-1,3-disulfonate). It is sometimes used as a counterstain, especially in fluorescence methods, to suppress background autofluorescence. It has functions as a histological stain, fluorescent dye, teratogen, and sodium channel blocker. It contains the Evans blue (4-) group.
An azo dye used to measure blood volume and cardiac output by dye dilution. It is highly soluble in water, has a strong binding affinity to plasma albumin, and disappears very slowly. See also: Evans blue (note moved to). Evans blue is a water-soluble azo dye, mainly used as a biological tracer in biomedical research, and currently has no clinical therapeutic applications [1]. - Its core application mechanism relies on its high binding affinity to albumin and its inability to cross intact biological barriers, thereby enabling the assessment of barrier integrity (e.g., blood-brain barrier, vascular endothelium, intestinal epithelium)[1]. - Evans blue is also used to map tissue perfusion, detect tumor angiogenesis, and evaluate the efficacy of barrier-protective drugs in preclinical models[1]. - In blood-brain barrier studies, Evans blue is considered the gold standard tracer for quantifying barrier disruption because its brain permeability directly reflects the severity of blood-brain barrier leakage[2]. |
| Molecular Formula |
C34H24N6O14S4.4NA
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| Molecular Weight |
960.81
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| Exact Mass |
959.982
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| CAS # |
314-13-6
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| Related CAS # |
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| PubChem CID |
9409
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| Appearance |
White to off-white solid powder
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| LogP |
10.785
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
20
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
62
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| Complexity |
1790
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
ATNOAWAQFYGAOY-UHFFFAOYSA-J
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| InChi Code |
InChI=1S/C34H28N6O14S4.4Na/c1-15-11-17(3-7-21(15)37-39-23-9-5-19-25(55(43,44)45)13-27(57(49,50)51)31(35)29(19)33(23)41)18-4-8-22(16(2)12-18)38-40-24-10-6-20-26(56(46,47)48)14-28(58(52,53)54)32(36)30(20)34(24)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
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| Chemical Name |
tetrasodium;4-amino-6-[[4-[4-[(8-amino-1-hydroxy-5,7-disulfonatonaphthalen-2-yl)diazenyl]-3-methylphenyl]-2-methylphenyl]diazenyl]-5-hydroxynaphthalene-1,3-disulfonate
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
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, avoid exposure to moisture. |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (2.60 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (2.60 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. View More
Solubility in Formulation 3: 2.5 mg/mL (2.60 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.0408 mL | 5.2039 mL | 10.4079 mL | |
| 5 mM | 0.2082 mL | 1.0408 mL | 2.0816 mL | |
| 10 mM | 0.1041 mL | 0.5204 mL | 1.0408 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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