superoxide indicator; blue-fluorescence dye

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.

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Dihydroethidium reacts with superoxide (O₂•⁻) with a rate constant of ~10⁵–10⁶ M⁻¹·s⁻¹, forming a specific hydroxylated product, 2‑OH‑E⁺. Other oxidants (peroxynitrite‑derived radicals, hydroxyl radical, perferryl iron) react with HE to form ethidium (E⁺) and non‑fluorescent dimeric products (e.g., E⁺‑E⁺) but do not yield 2‑OH‑E⁺. [1]
In cell‑free systems using hypoxanthine/xanthine oxidase (generating O₂•⁻) and DPTA‑NONOate (generating •NO), co‑generation of •NO and O₂•⁻ (forming ONOO⁻) inhibited 2‑OH‑E⁺ production and increased E⁺ and dimeric products in a concentration‑dependent manner. Fluorescence intensity from HE oxidation was not diminished by increasing •NO flux despite the decrease in 2‑OH‑E⁺, due to formation of E⁺ which has similar fluorescence spectrum. [1]

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.

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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Enzyme Assay: Not applicable (HE is a probe, not an enzyme substrate). However, the following method is described for detecting O₂•⁻ and other oxidants using HE: HE oxidation was monitored in a 96‑well fluorescence plate reader (excitation 485 nm, emission 595 nm) in phosphate buffer (50 mM, pH 7.4, containing 0.1 mM DTPA). Varying fluxes of •NO (from DPTA‑NONOate decomposition) and O₂•⁻ (from hypoxanthine/xanthine oxidase) were co‑generated. The rates of increase in fluorescence intensity were plotted against •NO and O₂•⁻ fluxes. HPLC analysis was performed using a fused core C18 column (2.6 µm, 100×4.6 mm) with gradient elution (aqueous mobile phase with increasing acetonitrile from 10–20% to 100% over 5 min, 0.1% TFA, flow rate 1.5 ml/min). Products (HE, 2‑OH‑E⁺, E⁺, and E⁺‑E⁺) were quantified using standards. [1]

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

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Cell Assay: RAW 264.7 macrophages were seeded in 96‑well plates, stimulated with phorbol 12‑myristate 13‑acetate (PMA, 200 ng/ml) in the presence of dihydroethidium (concentration not specified, but typically 10 μM from other studies). Fluorescence increase was monitored in a plate reader (excitation 485 nm, emission 595 nm) at 37 °C. Superoxide dismutase (SOD) but not catalase inhibited the fluorescence increase, indicating O₂•⁻‑dependent formation of 2‑OH‑E⁺. L‑NAME did not inhibit. Co‑stimulation with LPS (0.5 μg/ml), IFN‑γ (50 units/ml), and PMA decreased intracellular 2‑OH‑E⁺ and increased dimeric products. After 1 h incubation, cells were lysed and media collected; products were analyzed by HPLC as described above. [1]

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.

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[1]. Global profiling of reactive oxygen and nitrogen species in biological systems: high-throughput real-time analyses. J Biol Chem. 2012 Jan 27;287(5):2984-95.

See also: Hydroacetidine (note moved to).

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Dihydroethidium (hydroethidine) is a widely used fluorescent probe for superoxide detection. Its reaction with superoxide yields 2‑hydroxyethidium (2‑OH‑E⁺), a specific marker product. However, fluorescence measurements alone cannot distinguish 2‑OH‑E⁺ from ethidium (E⁺) due to spectral overlap; HPLC analysis is essential for proper quantification. Other oxidants (e.g., peroxynitrite, hydroxyl radical) produce E⁺ and dimers but not 2‑OH‑E⁺. The rate constant of HE with O₂•⁻ is ~10⁵–10⁶ M⁻¹·s⁻¹, which is much slower than the reaction of O₂•⁻ with SOD (~10⁹ M⁻¹·s⁻¹), so SOD can effectively compete. HE‑derived radicals do not react with molecular oxygen, preventing artifactual generation of O₂•⁻ and H₂O₂. The mitochondria‑targeted analog MitoSOX Red works similarly. Extraneous factors (light, sonication) can induce formation of both 2‑OH‑E⁺ and E⁺. [1]

C21H21N3

315.41

315.173

C, 79.97; H, 6.71; N, 13.32

104821-25-2

128682

Pale purple to light pink solid powder

1.2±0.1 g/cm3

580.4±50.0 °C at 760 mmHg

299.5±24.9 °C

0.0±1.6 mmHg at 25°C

1.680

3.06

2

3

2

24

419

0

NC1=CC=C2C3=C(C=C(N)C=C3)C(C4=CC=CC=C4)N(CC)C2=C1

XYJODUBPWNZLML-UHFFFAOYSA-N

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

5-ethyl-6-phenyl-6H-phenanthridine-3,8-diamine

2934.99.9001

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.

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

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.

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