Dihydroethidium (Hydroethidine)
(Synonyms: DHE) 目录号 : GC30025
Dihydroethidium（羟乙啶）（DHE）的氧化常被用作监测细胞产生的“活性氧（ROS）”的方法。
Cas No.:104821-25-2,38483-26-0
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
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- Purity: >98.00%
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本方案仅提供一个指导，请根据您的具体需要进行修改。
1. 制备染色液
(1)配置储存液:使用DMSO溶解Dihydroethidium，配置浓度为1-10mM的储存液。
注意:
① 未使用的储存液分装后在-20℃或-80°C避光保存，避免反复冻融；
② 本品易氧化，保存过程中尽量避免接触空气，需保存在氮气或氩气下，特别是溶液。
(2)配置工作液:用合适的缓冲液(如:无血清培养基或PBS)稀释储存液，配制浓度为1-10μM的工作液。
注意:请根据实际情况调整工作液浓度，现用现配。
2.细胞悬浮染色
(1)悬浮细胞：经4°C、1000g离心3-5分钟，弃去上清液，用PBS清洗两次，每次5分钟。
(2)贴壁细胞：使用PBS清洗两次，加入胰酶消化细胞，消化完成后经1000g离心3-5min。
(3)加入Dihydroethidium工作溶液重悬细胞，室温避光孵育5-30分钟。不同细胞最佳孵育时间不同，请根据具体实验需求自行摸索。
(4)孵育结束后，经1000g离心5分钟，去除上清液，加入PBS清洗2-3次，每次5分钟。
(5)用预温的无血清细胞培养基或PBS重悬细胞，通过荧光显微镜或流式细胞术观察。
3.细胞贴壁染色
(1)在无菌盖玻片上培养贴壁细胞。
(2)从培养基中移走盖玻片，吸出过量的培养基，将盖玻片放在潮湿的环境中。
(3)从盖玻片的一角加入100μL的染料工作液，轻轻晃动使染料均匀覆盖所有细胞。
(4) 室温避光孵育5-30分钟。不同细胞最佳孵育时间不同，请根据具体实验需求自行摸索。
(5)孵育结束后吸弃染料工作液，使用预温的培养液清洗盖玻片2~3次。
4.显微镜检测：Dihydroethidium的最大激发/发射波长为518/616nm。
注意事项：
① Dihydroethidium对光非常敏感，请尽量注意避光，以减缓荧光淬灭；
② Dihydroethidium氧化后产生乙锭类化合物，毒性较大，为了您的安全和健康，请穿实验服并戴一次性手套操作。
Dihydroethdium(Hydroethidine) (DHE) oxidation is commonly used as a method for monitoring cellular production of "reactive oxygen species (ROS)". Usually changes in DHE florescence due to oxidation in cells and tissues are measured by microscopy, flow cytometry and occassionaly by HPLC analysis [1]. Dihydroethdium is a hydrophobic uncharged compound that is able to cross extra- and intracellular membranes and, upon oxidation, becomes positively charged and accumulates in cells by intercalating into DNA, primarily by electrostatic interactions with DNA phosphate groups and further via hydrophobic interactions [2]. Its oxidation by different oxidizing systems has been used increasingly for fluorescent analysis of ROS output in cells and tissues. Dihydroethdium -derived red fluorescence observed with rhodamine filter (excitation 490; emission 590 nm) was attributed to ethidium compound formation, a two-electron oxidation product, and the red fluorescence was obtained more specifically with superoxide-generating systems (xanthine or glucose oxidase) rather than with oxidants such as hydrogen peroxide, peroxynitrite, or hydroxyl radical (generated by the Fenton reaction) [3,4].
Detection of ROS production in the liver tissues [5]
The levels of ROS production in the liver tissues were determined by dihydroethidium (DHE) staining. Frozen sections of liver tissues in each group (5-μm-thick) were prepared and incubated with DHE (7.5 mM,) for 30 min in the dark at 37 °C. After staining with DAPI, the sections were observed using a fluorescence microscope.
References:
[1]. Wagner B A, Buettner G R. Quantitative Changes in Dihydroethidium (DHE) Oxidation Products from Isolated Mitochondria While Respiring on Select Substrates and the Effects Mitochondrial Inhibitors Commonly Used in Bioenergetic Profiling[J]. Free Radical Biology and Medicine, 2016, 100: S31.
[2]. Garbett N C, Hammond N B, Graves D E. Influence of the amino substituents in the interaction of ethidium bromide with DNA[J]. Biophysical journal, 2004, 87(6): 3974-3981.
[3]. Benov L, Sztejnberg L, Fridovich I. Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical[J]. Free Radical Biology and Medicine, 1998, 25(7): 826-831.
[4]. Biemond P, Swaak A J G, Beindorff C M, et al. Superoxide-dependent and-independent mechanisms of iron mobilization from ferritin by xanthine oxidase. Implications for oxygen-free-radical-induced tissue destruction during ischaemia and inflammation[J]. Biochemical Journal, 1986, 239(1): 169-173.
[5]. Zheng J, Chen L, Lu T, et al. MSCs ameliorate hepatocellular apoptosis mediated by PINK1-dependent mitophagy in liver ischemia/reperfusion injury through AMPKα activation[J]. Cell death & disease, 2020, 11(4): 1-19.
Dihydroethidium（羟乙啶）（DHE）的氧化常被用作监测细胞产生的“活性氧（ROS）”的方法。通常使用显微镜、流式细胞术和偶尔的高效液相色谱分析来测量细胞和组织中由于氧化而引起的DHE荧光变化[1]。Dihydroethidium是一种亲水性、不带电的化合物,能够穿越细胞内外膜,在氧化后变成带正电的形式,并通过静电相互作用和疏水相互作用,通过与DNA磷酸根团结合而积聚在细胞中[2]。它在不同的氧化系统中的氧化已越来越多地用于细胞和组织中ROS产生的荧光分析。使用罗丹明滤光片（激发波长490纳米；发射波长590纳米）观察到的Dihydroethidium衍生的红色荧光被归因于乙啶化合物的形成,这是一种两电子氧化产物,并且红色荧光与产生超氧化物的系统（黄嘌呤氧化酶或葡萄糖氧化酶）相比,与氢过氧化物、过硝酸盐或羟基自由基（由Fenton反应产生）等氧化剂获得更加明显[3,4]。
检测肝组织中的ROS产生[5]
使用Dihydroethidium（DHE）染色确定肝组织中ROS的产生水平。每组制备肝组织的冰冻切片（厚度为5微米）,在暗处37°C下与DHE（7.5毫摩尔）孵育30分钟。染色后用DAPI染色,使用荧光显微镜观察切片。
| Cas No. | 104821-25-2,38483-26-0 | SDF | |
| 别名 | DHE | ||
| Canonical SMILES | NC1=CC=C2C3=C(C=C(N)C=C3)C(C4=CC=CC=C4)N(CC)C2=C1 | ||
| 分子式 | C21H21N3 | 分子量 | 315.41 |
| 溶解度 | DMSO : ≥ 50 mg/mL (158.52 mM);Water : < 0.1 mg/mL (insoluble) | 储存条件 | Store at -20°C, protect from light |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
 |
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 | 6.341 mL |
| 10 mM | 0.317 mL | 1.5852 mL | 3.1705 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: another inconvenient truth
Free Radic Biol Med 2010 Apr 15;48(8):983-1001.20116425 PMC3587154
Hydroethidine (HE; or Dihydroethidium) is the most popular fluorogenic probe used for detecting intracellular superoxide radical anion. The reaction between superoxide and HE generates a highly specific red fluorescent product, 2-hydroxyethidium (2-OH-E(+)). In biological systems, another red fluorescent product, ethidium, is also formed, usually at a much higher concentration than 2-OH-E(+). In this article, we review the methods to selectively detect the superoxide-specific product (2-OH-E(+)) and the factors affecting its levels in cellular and biological systems. The most important conclusion of this review is that it is nearly impossible to assess the intracellular levels of the superoxide-specific product, 2-OH-E(+), using confocal microscopy or other fluorescence-based microscopic assays and that it is essential to measure by HPLC the intracellular HE and other oxidation products of HE, in addition to 2-OH-E(+), to fully understand the origin of red fluorescence. The chemical reactivity of mitochondria-targeted Hydroethidine (Mito-HE, MitoSOX red) with superoxide is similar to the reactivity of HE with superoxide, and therefore, all of the limitations attributed to the HE assay are applicable to Mito-HE (or MitoSOX) as well.
HPLC-based monitoring of products formed from hydroethidine-based fluorogenic probes--the ultimate approach for intra- and extracellular superoxide detection
Biochim Biophys Acta 2014 Feb;1840(2):739-44.23668959 PMC3858408
Background: Nearly ten years ago, we demonstrated that superoxide radical anion (O2⋅¿ reacts with the Hydroethidine dye (HE, also known as Dihydroethidium, DHE) to form a diagnostic marker product, 2-hydroxyethidium (2-OH-E(+)). This particular product is not derived from reacting HE with other biologically relevant oxidants (hydrogen peroxide, hydroxyl radical, or peroxynitrite). This discovery negated the longstanding view that O2⋅¿reacts with HE to form the other oxidation product, ethidium (E(+)). It became clear that due to the overlapping fluorescence spectra of E(+) and 2-OH-E(+), fluorescence-based techniques using the "red fluorescence" are not suitable for detecting and measuring O2⋅¿in cells using HE or other structurally analogous fluorogenic probes (MitoSOX(TM) Red or hydropropidine). However, using HPLC-based assays, 2-OH-E(+) and analogous hydroxylated products can be easily detected and quickly separated from other oxidation products. Scope of review: The principles discussed in this chapter are generally applicable in free radical biology and medicine, redox biology, and clinical and translational research. The assays developed here could be used to discover new and targeted inhibitors for various superoxide-producing enzymes, including NADPH oxidase (NOX) isoforms. Major conclusions: HPLC-based approaches using site-specific HE-based fluorogenic probes are eminently suitable for monitoring O2⋅¿in intra- and extracellular compartments and in mitochondria. The use of fluorescence-microscopic methods should be avoided because of spectral overlapping characteristics of O2⋅¿derived marker product and other, non-specific oxidized fluorescent products formed from these probes. General significance: Methodologies and site-specific fluorescent probes described in this review can be suitably employed to delineate oxy radical dependent mechanisms in cells under physiological and pathological conditions. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.
On the use of fluorescence lifetime imaging and Dihydroethidium to detect superoxide in intact animals and ex vivo tissues: a reassessment
Free Radic Biol Med 2014 Feb;67:278-84.24200598 PMC4275029
Recently, D.J. Hall et al. reported that ethidium (E(+)) is formed as a major product of Hydroethidine (HE) or Dihydroethidium reaction with superoxide (O2(-)) in intact animals with low tissue oxygen levels (J. Cereb. Blood Flow Metab. 32:23-32, 2012). The authors concluded that measurement of E(+) is an indicator of O2(-) formation in intact brains of animals. This finding is in stark contrast to previous reports using in vitro systems showing that 2-hydroxyethidium, not ethidium, is formed from the reaction between O2(-) and HE. Published in vivo results support the in vitro findings. In this study, we performed additional experiments in which HE oxidation products were monitored under different fluxes of O2(-). Results from these experiments further reaffirm our earlier findings (H. Zhao et al., Free Radic. Biol. Med. 34:1359, 2003). We conclude that whether in vitro or in vivo, E(+) measured by HPLC or by fluorescence lifetime imaging is not a diagnostic marker product for O2(-) reaction with HE.
Assessment of myeloperoxidase activity by the conversion of Hydroethidine to 2-chloroethidium
J Biol Chem 2014 Feb 28;289(9):5580-95.24436331 PMC3937635
Oxidants derived from myeloperoxidase (MPO) contribute to inflammatory diseases. In vivo MPO activity is commonly assessed by the accumulation of 3-chlorotyrosine (3-Cl-Tyr), although 3-Cl-Tyr is formed at low yield and is subject to metabolism. Here we show that MPO activity can be assessed using Hydroethidine (HE), a probe commonly employed for the detection of superoxide. Using LC/MS/MS, (1)H NMR, and two-dimensional NOESY, we identified 2-chloroethidium (2-Cl-E(+)) as a specific product when HE was exposed to hypochlorous acid (HOCl), chloramines, MPO/H2O2/chloride, and activated human neutrophils. The rate constant for HOCl-mediated conversion of HE to 2-Cl-E(+) was estimated to be 1.5 × 10(5) M(-1)s(-1). To investigate the utility of 2-Cl-E(+) to assess MPO activity in vivo, HE was injected into wild-type and MPO-deficient (Mpo(-/-)) mice with established peritonitis or localized arterial inflammation, and tissue levels of 2-Cl-E(+) and 3-Cl-Tyr were then determined by LC/MS/MS. In wild-type mice, 2-Cl-E(+) and 3-Cl-Tyr were detected readily in the peritonitis model, whereas in the arterial inflammation model 2-Cl-E(+) was present at comparatively lower concentrations (17 versus 0.3 pmol/mg of protein), and 3-Cl-Tyr could not be detected. Similar to the situation with 3-Cl-Tyr, tissue levels of 2-Cl-E(+) were decreased substantially in Mpo(-/-) mice, indicative of the specificity of the assay. In the arterial inflammation model, 2-Cl-E(+) was absent from non-inflamed arteries and blood, suggesting that HE oxidation occurred locally in the inflamed artery. Our data suggest that the conversion of exogenous HE to 2-Cl-E(+) may be a useful selective and sensitive marker for MPO activity in addition to 3-Cl-Tyr.
Superoxide reacts with Hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide
Free Radic Biol Med 2003 Jun 1;34(11):1359-68.12757846 10.1016/s0891-5849(03)00142-4
Hydroethidine (HE) or Dihydroethidium (DHE), a redox-sensitive probe, has been widely used to detect intracellular superoxide anion. It is a common assumption that the reaction between superoxide and HE results in the formation of a two-electron oxidized product, ethidium (E+), which binds to DNA and leads to the enhancement of fluorescence (excitation, 500-530 nm; emission, 590-620 nm). However, the mechanism of oxidation of HE by the superoxide anion still remains unclear. In the present study, we show that superoxide generated in several enzymatic or chemical systems (e.g., xanthine/xanthine oxidase, endothelial nitric oxide synthase, or potassium superoxide) oxidizes HE to a fluorescent product (excitation, 480 nm; emission, 567 nm) that is totally different from E+. HPLC measurements revealed that the HE/superoxide reaction product elutes differently from E+. This new product exhibited an increase in fluorescence in the presence of DNA. Mass spectral data indicated that the molecular weight of the HE/superoxide reaction product is 330, while ethidium has a molecular weight of 314. We conclude that the reaction between superoxide and HE forms a fluorescent marker product that is different from ethidium. Potential implications of this finding in intracellular detection and imaging of superoxide are discussed.