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Purity: ≥98%
Dihydroethidium (formerly known as Hydroethidine and PD-MY 003) is a cell-permeable blue fluorescent dye and a superoxide indicator which exhibits blue-fluorescence in the cytosol until oxidized, where it intercalates within the cell's DNA, staining its nucleus with a bright fluorescent red. DHE-derived fluorescence at 570 nm is a convenient method to detect intracellular and extracellular superoxide produced by phagocytic and vascular NADPH oxidase. In addition, Dihydroethidium is also reported to be neuroprotective by reducing superoxide in mice after stroke.
| Targets |
superoxide indicator; blue-fluorescence dye
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|---|---|
| ln Vitro |
General procedure:
Preparation of dihydroethidium working solution 1.1 Prepare the stock solution by dissolving 1 mg of dihydroethidium in 0.31 mL DMSO to make 10 mM stock solution. Note: Avoid repeated freezing and thawing cycle, it is advised that the stock solution be stored at -20°C or -80°C and away from light. 1.2 Preparation of dihydroethidium working solution: Dilute the stock solution into 1–10 μM working solution in PBS or serum-free cell culture medium. Note: You may adjust the concentration of Dihydroethidium working solution based on your specific needs. Cell staining 2.1: Suspension cells: Centrifuge 1000g of suspended cells for 3–5 minutes at 4°C; discard supernatant. Use PBS to wash twice, for five minutes each time. Adherent cells: To isolate the cells and create a single cell suspension, discard the cell culture medium and add trypsin. Discard the supernatant after centrifuging at 1000g for three to five minutes at 4°C. Use PBS to wash twice, for five minutes each time. 2.2 Add 1 mL of the Dihydroethidium working solution, then let it sit at room temperature for half an hour. 2.3 Discard the supernatant after centrifuging at 400 g for three to four minutes at 4°C. 2.4 Wash the cells with PBS twice, five minutes/each time. 2.5 Re-suspend the cells in PBS or serum-free cell culture medium, then use a flow cytometer or fluorescence microscope to observe them. Storage for a year at -20°C with protection from light. Precautions: 1. The original solution should be kept refrigerated at -20°C or -80°C, shielded from light, and avoid freezing and thaw too frequently. 2. You may adjust the concentration of Dihydroethidium working solution based on your specific needs. 3. This product is intended for research use only. 4. Please wear a lab coat and disposable gloves for your own health and safety when operating. |
| ln Vivo |
Oxidative stress-related indicators in OXA-induced HSOS in mice[2]
The microarray results suggested that oxidative stress may serve an important role in OXA-induced HSOS. Therefore, the levels of common oxidative stress markers were determined and changes in ROS were observed by DHE staining. MDA is a lipid peroxidation product in vivo, which can cause cytotoxicity. The MDA levels in the mice livers were significantly increased following the administration of 10 mg/kg OXA (P<0.05; Fig. 7A). SOD is an antioxidant metal enzyme that can catalyze the superoxide anion radical and provide cellular defenses against ROS. CAT is also an antioxidant enzyme that protects cells from the toxicity of H2O2. GSH has antioxidant and integrative detoxification effects. OXA markedly reduced the levels of SOD, CAT and GSH in the mice livers (P<0.05; Fig. 7B-D). Furthermore, DHE probe technology was used to analyze changes in ROS levels. The results showed that the ROS levels in the livers of the mice in the OXA group were increased in a dose-dependent manner (Fig. 7E). Taken together, these data confirmed that oxidative stress may have an important role in the liver damage of mice with OXA-induced HSOS. |
| Enzyme Assay |
Hydroethidine (HE) or dihydroethidium and its mitochondria-targeted analog conjugated to a triphenylphosphonium moiety (MitoSOXTM Red) react rapidly with superoxide (k ∼106 m−1 s−1), forming a specific hydroxylated marker product, 2-hydroxyethidium (2-OH-E+) or 2-hydroxymitoethidium (2-OH-Mito-E+)[1].
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| Cell Assay |
Intracellular ROS measurements[3]
Two dyes were used to detect ROS: 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA: excitation wavelength, 504 nm; emission wavelength, 524 nm) and dihydroethidium (DHE; excitation wavelength, 518 nm; emission wavelength, 605 nm). Both dyes were reconstituted with DMSO. Monocytes and THP-1 cells were incubated with dye at 37°C for 30 min. Cell pellets were resuspended in media and incubated for 15 min at 37°C to allow esterases to cleave CM-H2DCFDA to trap it inside the cell. Cells were then incubated with a2NTD, PMA, or media alone with or without 100 μM diethyldithiocarbamic acid sodium salt trihydrate. Two methods were used to detect intracellular ROS. After a2NTD stimulation, cells were analyzed for fluorescence via flow cytometer on an LSR II or via a microplate reader in a 96-well black plate |
| Animal Protocol |
Staining of dihydroethidium (DHE)-reactive oxygen species (ROS)[2]
Frozen liver sections (−26°C; 6 µm thickness) were warmed at room temperature and mounted with an anti-fluorescence quenching solution for 5 min. ROS dye solution was added dropwise and sections were incubated for 30 min at 37°C in the dark. Subsequently, the sections were washed three times with phosphate-buffered saline (PBS, pH 7.4) and DAPI staining solution was added dropwise and incubated for 10 min at room temperature in the dark. After washing three times with PBS and drying, the sections were mounted with an anti-fluorescence quenching solution. The sections were observed under a fluorescence microscope and images were captured. |
| References | |
| Additional Infomation |
See also: Hydroethidine (annotation moved to).
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| Molecular Formula |
C21H21N3
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|---|---|---|
| Molecular Weight |
315.41
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| Exact Mass |
315.173
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| Elemental Analysis |
C, 79.97; H, 6.71; N, 13.32
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| CAS # |
104821-25-2
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| Related CAS # |
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| PubChem CID |
128682
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| Appearance |
Pale purple to light pink solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
580.4±50.0 °C at 760 mmHg
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| Flash Point |
299.5±24.9 °C
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| Vapour Pressure |
0.0±1.6 mmHg at 25°C
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| Index of Refraction |
1.680
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| LogP |
3.06
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
24
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| Complexity |
419
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| Defined Atom Stereocenter Count |
0
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| SMILES |
NC1=CC=C2C3=C(C=C(N)C=C3)C(C4=CC=CC=C4)N(CC)C2=C1
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| InChi Key |
XYJODUBPWNZLML-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H21N3/c1-2-24-20-13-16(23)9-11-18(20)17-10-8-15(22)12-19(17)21(24)14-6-4-3-5-7-14/h3-13,21H,2,22-23H2,1H3
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| Chemical Name |
5-ethyl-6-phenyl-6H-phenanthridine-3,8-diamine
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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: This product requires protection from light (avoid light exposure) during transportation and storage. |
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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 (7.93 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.1705 mL | 15.8524 mL | 31.7048 mL | |
| 5 mM | 0.6341 mL | 3.1705 mL | 6.3410 mL | |
| 10 mM | 0.3170 mL | 1.5852 mL | 3.1705 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.
 Oxidation of HE by O2˙̄,•NO, and co-generated•NO and O2˙̄and global profiling of products.J Biol Chem.2012 Jan 27;287(5):2984-95. |
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 Real-time monitoring of products of HE oxidation by activated macrophages.J Biol Chem.2012 Jan 27;287(5):2984-95. |
 Global profiling of H2O2and ONOO−-derived oxidants by monitoring the oxidation of Amplex® Red.J Biol Chem.2012 Jan 27;287(5):2984-95. |
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