Hydroethidine [Dihydroethidium] *CAS 104821-25-2*
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Physical properties
Molecular weight | 315.41 |
Solvent | DMSO |
Spectral properties
Excitation (nm) | 500 |
Emission (nm) | 582 |
Storage, safety and handling
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
Storage | Freeze (< -15 °C); Minimize light exposure |
UNSPSC | 12352200 |
Overview | SDSProtocol |
See also: Reactive Oxygen Species (ROS)
CAS 104821-25-2 | Molecular weight 315.41 | Excitation (nm) 500 | Emission (nm) 582 |
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.
Platform
Fluorescence microplate reader
Excitation | 520 nm |
Emission | 600 nm |
Cutoff | 550 nm |
Recommended plate | Solid black |
Example protocol
PREPARATION OF STOCK SOLUTION
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.
PREPARATION OF WORKING SOLUTION
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.
SAMPLE EXPERIMENTAL PROTOCOL
- 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.
- Determine the baseline fluorescence intensity of a sample of the loaded cells prior to exposing the cells to experimental inducements.
- Negative controls should be assessed as follows:
- 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.
- 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.
- 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.
- 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).
Calculators
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 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 317.048 µL | 1.585 mL | 3.17 mL | 15.852 mL | 31.705 mL |
5 mM | 63.41 µL | 317.048 µL | 634.095 µL | 3.17 mL | 6.341 mL |
10 mM | 31.705 µL | 158.524 µL | 317.048 µL | 1.585 mL | 3.17 mL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
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Citations
View all 16 citations: Citation Explorer
Redox-related Molecular Mechanism of Sensitizing Colon Cancer Cells to Camptothecin Analog SN38
Authors: Nikolova, Biliana and Semkova, Severina and Tsoneva, Iana and Stoyanova, Elena and Lefterov, Pavel and Lazarova, Dessislava and Zhelev, Zhivko and Aoki, Ichio and Higashi, Tatsuya and Bakalova, Rumiana
Journal: Anticancer Research (2020): 5159--5170
Authors: Nikolova, Biliana and Semkova, Severina and Tsoneva, Iana and Stoyanova, Elena and Lefterov, Pavel and Lazarova, Dessislava and Zhelev, Zhivko and Aoki, Ichio and Higashi, Tatsuya and Bakalova, Rumiana
Journal: Anticancer Research (2020): 5159--5170
Bifunctional nanozyme activities of layered double hydroxide derived Co-Al-Ce mixed metal oxides for antibacterial application
Authors: Chen, Chao and Wang, Yi and Zhang, Dun
Journal: Journal of Oceanology and Limnology (2020): 1--13
Authors: Chen, Chao and Wang, Yi and Zhang, Dun
Journal: Journal of Oceanology and Limnology (2020): 1--13
Dual response mimetic enzyme of novel Co4S3/Co3O4 composite nanotube for antibacterial application
Authors: Wang, Jin and Wang, Yi and Zhang, Dun and Xu, Chaojie and Xing, Ronge
Journal: Journal of Hazardous Materials (2020): 122278
Authors: Wang, Jin and Wang, Yi and Zhang, Dun and Xu, Chaojie and Xing, Ronge
Journal: Journal of Hazardous Materials (2020): 122278
Exploring the bactericidal performance and application of novel mimic enzyme Co4S3
Authors: Wang, Jin and Wang, Yi and Zhang, Dun
Journal: Journal of colloid and interface science (2019)
Authors: Wang, Jin and Wang, Yi and Zhang, Dun
Journal: Journal of colloid and interface science (2019)
Bifunctionalized Novel Co-V MMO Nanowires: Intrinsic Oxidase and Peroxidase Like Catalytic Activities for Antibacterial Application
Authors: Wang, Yi and Chen, Chao and Zhang, Dun and Wang, Jin
Journal: Applied Catalysis B: Environmental (2019): 118256
Authors: Wang, Yi and Chen, Chao and Zhang, Dun and Wang, Jin
Journal: Applied Catalysis B: Environmental (2019): 118256
Anthocyanin-rich bilberry extract induces apoptosis in acute lymphoblastic leukemia cells via redox-sensitive epigenetic modifications
Authors: León-González, Antonio J and Sharif, Tanveer and Auger, Cyril and Abbas, Malak and Fuhrmann, Guy and Schini-Kerth, Valérie B
Journal: Journal of Functional Foods (2018): 227--234
Authors: León-González, Antonio J and Sharif, Tanveer and Auger, Cyril and Abbas, Malak and Fuhrmann, Guy and Schini-Kerth, Valérie B
Journal: Journal of Functional Foods (2018): 227--234
Tumor suppressor NPRL2 induces ROS production and DNA damage response
Authors: Ma, Yinxing and Silveri, Licia and LaCava, John and Dokudovskaya, Svetlana
Journal: Scientific reports (2017): 1--15
Authors: Ma, Yinxing and Silveri, Licia and LaCava, John and Dokudovskaya, Svetlana
Journal: Scientific reports (2017): 1--15
Role of epigenetic regulation on the induction of apoptosis in Jurkat leukemia cells by white grape pomace rich in phenolic compounds
Authors: Le{\'o}n-Gonz{\'a}lez, Antonio J and Jara-Palacios, M Jos{\'e} and Abbas, Malak and Heredia, Francisco J and Schini-Kerth, Val{\'e}rie B
Journal: Food \& function (2017): 4062--4069
Authors: Le{\'o}n-Gonz{\'a}lez, Antonio J and Jara-Palacios, M Jos{\'e} and Abbas, Malak and Heredia, Francisco J and Schini-Kerth, Val{\'e}rie B
Journal: Food \& function (2017): 4062--4069
Role of epigenetic regulation on the induction of apoptosis in Jurkat leukemia cells by white grape pomace rich in phenolic compounds
Authors: León-González, Antonio J and Jara-Palacios, M José and Abbas, Malak and Heredia, FJ and Schini-Kerth, Valérie
Journal: Food & Function (2017)
Authors: León-González, Antonio J and Jara-Palacios, M José and Abbas, Malak and Heredia, FJ and Schini-Kerth, Valérie
Journal: Food & Function (2017)
Inhibitory effect of Sophora subprosrate polysaccharide on mitochondria oxidative stress induced by PCV-2 infection in RAW264. 7 cells
Authors: Su, Zi-Jie and Yang, Jian and Luo, Wen-Juan and Wei, Ying-Yi and Shuai, Xue-Hong and Hu, Ting-Jun
Journal: International Journal of Biological Macromolecules (2016)
Authors: Su, Zi-Jie and Yang, Jian and Luo, Wen-Juan and Wei, Ying-Yi and Shuai, Xue-Hong and Hu, Ting-Jun
Journal: International Journal of Biological Macromolecules (2016)
References
View all 24 references: Citation Explorer
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)
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)
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)
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)
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
Authors: Zielonka J, Vasquez-Vivar J, Kalyanaraman B.
Journal: Free Radic Biol Med (2006): 1050
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
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
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
Authors: Zielonka J, Zhao H, Xu Y, Kalyanaraman B.
Journal: Free Radic Biol Med (2005): 853
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
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
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
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
Authors: Papapostolou I, Patsoukis N, Georgiou CD.
Journal: Anal Biochem (2004): 290
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FAQ
What is the difference between MitoROS 580 and dihydroethidium?
Why should I use an absorbance ratio at A575nm/A605nm when using most of your Amplite® Colorimetric Assay Kits?
How should I reconstitute an NADPH standard?
How can I lyse my cells without lysing the nuclear membrane?
What are the differences between calcium ion indicators: Cal 520, Cal 520FF, and Cal 520N?
Why should I use an absorbance ratio at A575nm/A605nm when using most of your Amplite® Colorimetric Assay Kits?
How should I reconstitute an NADPH standard?
How can I lyse my cells without lysing the nuclear membrane?
What are the differences between calcium ion indicators: Cal 520, Cal 520FF, and Cal 520N?