Hydroethidine [Dihydroethidium] *CAS 104821-25-2*
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.
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
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Citations
View all 17 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
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Journal: Journal of Oceanology and Limnology (2020): 1--13
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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
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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)
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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
References
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
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)
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)
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
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