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Hydroethidine [Dihydroethidium] *CAS 104821-25-2*

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Physical properties
Molecular weight315.41
Spectral properties
Excitation (nm)500
Emission (nm)582
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure


Molecular weight
Excitation (nm)
Emission (nm)
Hydroethidine operates effectively as a probe for measurement of reactive oxygen species. The dye enters cells freely and is dehydrogenated to an ethidium compound. The probe has been used extensively with NK cell and as a vital dye for identification of proliferation and hypoxic cells in tumors. Studies have been performed using neutrophils and endothelial cells as well as HL60 cells and macrophages. A major advantage of this probe is its ability to distinguish between superoxide and H2O2. Fluorescence emission occurs at around 600 nm.


Fluorescence microplate reader

Excitation520 nm
Emission600 nm
Cutoff550 nm
Recommended plateSolid black

Example protocol


Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Hydroethidine [Dihydroethidium] stock solution:
Make 5-10 mM DMSO stock solution. Note: The unused DMSO stock solution should be aliquoted into single use vial and stored at -20 °C. Keep awayfrom light.


Hydroethidine [Dihydroethidium] dye working solution:
Make the dye working concentration of 5 – 20 µM in a physiological buffer (such as PBS, HBSS, HEPES). Note: The optimal working concentration for your application must be empirically determined.


  1. Add equal volume (such as 100 µL of the cells in growth medium) of the dye working solution to the cells, and incubate the cells at RT or 37°C for 5 to 60 minutes.

  2. Determine the baseline fluorescence intensity of a sample of the loaded cells prior to exposing the cells to experimental inducements.

  3. Negative controls should be assessed as follows:

    1. Examine the fluorescence of cell-free mixtures of dye and buffer/media with and without the inducer. In the absence of extracellular esterases and other oxidative enzymes, the gradual increase in fluorescence over time may be related to spontaneous hydrolysis, atmospheric oxidation, and/or light-induced oxidation.

    2. Examine the fluorescence of untreated (control) loaded cells that have been maintained in growth medium or buffer. In healthy cells, oxygen radicals are eliminated by cellular enzymes and/or natural antioxidants. Following the dye-loading recovery period, healthy cells should exhibit a low level of fluorescence that is relatively stable for the duration of the experiment; however, a gradual increase (due to auto-oxidation) or decrease (due to loss of dye from cells or photobleaching) in fluorescence may be observed. In the absence of any stimulus or inducement, a burst of fluorescence in healthy, untreated cells could indicate progress to cell death or some other oxidative event.

  4. Positive controls may be stimulated with tert-butyl hydroperoxide (TBHP) to a final concentration of ~100 µM (increase or decrease dose based on the sensitivity and response of the cells).


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Hydroethidine [Dihydroethidium] *CAS 104821-25-2* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM317.048 µL1.585 mL3.17 mL15.852 mL31.705 mL
5 mM63.41 µL317.048 µL634.095 µL3.17 mL6.341 mL
10 mM31.705 µL158.524 µL317.048 µL1.585 mL3.17 mL

Molarity calculator

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Spectral properties

Excitation (nm)500
Emission (nm)582



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View all 24 references: Citation Explorer
The confounding effects of light, sonication, and Mn(III)TBAP on quantitation of superoxide using hydroethidine
Authors: Zielonka J, Vasquez-Vivar J, Kalyanaraman B.
Journal: Free Radic Biol Med (2006): 1050
Pulse radiolysis and steady-state analyses of the reaction between hydroethidine and superoxide and other oxidants
Authors: Zielonka J, Sarna T, Roberts JE, Wishart JF, Kalyanaraman B.
Journal: Arch Biochem Biophys. (2006)
Generation of superoxide anion by equine spermatozoa as detected by dihydroethidium
Authors: Burnaugh L, Sabeur K, Ball BA.
Journal: Theriogenology. (2006)
Analysis of dihydroethidium-derived oxidation products by HPLC in the assessment of superoxide production and NADPH oxidase activity in vascular systems
Authors: Fern, undefined and es DC, Wosniak J, Pescatore LA, Bertoline MA, Liberman M, Laurindo F, Santos CX.
Journal: Am J Physiol Cell Physiol. (2006)
Mechanistic similarities between oxidation of hydroethidine by Fremy's salt and superoxide: stopped-flow optical and EPR studies
Authors: Zielonka J, Zhao H, Xu Y, Kalyanaraman B.
Journal: Free Radic Biol Med (2005): 853
Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence
Authors: Zhao H, Joseph J, Fales HM, Sokoloski EA, Levine RL, Vasquez-Vivar J, Kalyanaraman B.
Journal: Proc Natl Acad Sci U S A (2005): 5727
Interference of non-specific peroxidases in the fluorescence detection of superoxide radical by hydroethidine oxidation: a new assay for H2O2
Authors: Patsoukis N, Papapostolou I, Georgiou CD.
Journal: Anal Bioanal Chem (2005): 1065
Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay
Authors: Fink B, Laude K, McCann L, Doughan A, Harrison DG, Dikalov S.
Journal: Am J Physiol Cell Physiol (2004): C895
Double staining of Plasmodium falciparum nucleic acids with hydroethidine and thiazole orange for cell cycle stage analysis by flow cytometry
Authors: Jouin H, Daher W, Khalife J, Ricard I, Puijalon OM, Capron M, Dive D.
Journal: Cytometry A (2004): 34
The fluorescence detection of superoxide radical using hydroethidine could be complicated by the presence of heme proteins
Authors: Papapostolou I, Patsoukis N, Georgiou CD.
Journal: Anal Biochem (2004): 290