Background
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.
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。
参考文献:
[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.
Protocol
| Detection of intracellular ROS by flow cytometry [1]: |
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Cell lines
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Platelet isolated from Human blood
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Preparation Method
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Platelet suspensions were rested for 30 minutes at 37°C before incubation with 5 μM Dihydroethidium for 15 minutes. Platelets were then stimulated with the physiological stimuli thrombin (0.01–1.0 unit/ml), CRP (0.1–10 μg/ml), ADP (1–100 μM) or arachidonic acid (1–100 μM) for 15 minutes or with Aβ peptides (1–100 μM). Where indicated, a ROS scavenger (i.e. NAC) or an inhibitor (i.e. VAS2870 or indomethacin) was coincubated with stimuli. At the end of the incubation time, platelet suspensions were diluted 1:10 in cold modified Tyrode’s-HEPES buffer. Within 30 minutes from preparation, samples were then analyzed using a FACSAria III flow cytometer with 85 μm nozzle at low flow rate. Gate selection was performed using logarithmic side and forward scattering with the gain set at 40 and 220 mV, respectively. Fluorescence excitation was obtained with a violet laser at 405 nm and emission was collected at 590 nm with a LP600 filter. Baseline for unstimulated samples was set at 200 relative fluorescence units.
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Reaction Conditions
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5 μMfor 15 minutes
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Applications
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Dihydroethidium isdirectly oxidized to ethidium by other ROS, while only superoxide anion generates 2OH-Et+.Only 2OH-Et+ (but notethidium) has an excitation peak around 400 nm wavelength withemission at 580 nm.
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| Detection of intracellular ROS by confocal microscopy [1]: |
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Animal models
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Platelet isolated from Human blood
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Preparation Method
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Glass coverslips were coated with 0.1 mg/ml fibrillar collagen I from equine tendons (Horm collagen). Platelets were diluted to a density of 4 x 107 /ml and treated with 10 μM Dihydroethidium. After 1 minute of incubation, platelets were dispensed over the coverslip in a live cell-imaging chamber. Where indicated, ROS scavenger N-acetylcysteine (NAC, 1 mM) or NOX inhibitor VAS2870 (10 μM) were added to the live cell chamber. Oxidation-dependent probe conversion and fluorescence generation were monitored by confocal imaging for a period of 10 min (405/580 nm ex/em). Images were collected every 10 seconds using a Zeiss 510 LSM confocal microscope equipped with a 40x oil immersion lens and single cell fluorescence was quantified using Zeiss LSM Examiner software.
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Dosage form
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10 μMfor 1 minute
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Applications
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Concentrations of thrombin from 0.1unit/ml, CRP from 3μg/ml and arachidonic acid from 30 μMsignificantly increased fluorescence evels, while ADP did not significantly increase 2OH-Et+ generation.
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参考文献:
[1]: Abubaker A A, Vara D, Eggleston I, et al. A novel flow cytometry assay using dihydroethidium as redox-sensitive probe reveals NADPH oxidase-dependent generation of superoxide anion in human platelets exposed to amyloid peptide β[J]. Platelets, 2019, 30(2): 181-189.
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