Dihydroethidium (DHE) 二氢乙锭

搜索关键词：

Superoxide (O2-·) 超氧化物阴离子;Reactive oxygen species (ROS) 活性氧；H2DCFDA (DCFH-DA, DCFH)；吞噬细胞呼吸爆发 Respiratory Burst; Mitosox Red（M36008, Invitrogen）; CAS：104821-25-2

产品信息

产品描述

二氢乙锭（Dihydroethidium，DHE），也称为Hydroethidine，是一种最常用的超氧化物阴离子荧光检测探针。DHE本身是一种细胞膜渗透性的蓝色探针（λ Ex/λ Em: 370/420nm），一旦进入细胞后与超氧化物阴离子反应，生成2-hydroxyethidium。2-hydroxyethidium插入核酸后产生红色荧光（λ Ex/λ Em: 535/610 nm），能够被荧光显微镜、流式细胞仪或其他合适仪器检测。作为一种选择性且灵敏的荧光探针，DHE能用来检测吞噬细胞呼吸爆发中的活性氧（ROS）水平，也能用来检测培养细胞中的超氧化物阴离子水平。DHE也能用作活体染料，流式检测中用于完整细胞的成像和功能分析。还能用在各种凋亡模型中观察荧光的增强。另外，DHE也能用于活细胞线粒体超氧化物生成的检测，虽然其结构类似物MitoSOX Red具有更加特异性的线粒体定位能力。通常情况30~40min内能得到均匀标记的细胞。

产品特性

1）CAS NO.：104821-25-2

2）化学名：5-ethyl-5,6-dihydro-6-phenyl-3,8-phenanthridinediamine，3,8-Diamino-5,6-dihydro-5-ethyl-6- phenylphenanthridine，2,7-Diamino-10-ethyl-9-phenyl-9,10-dihydrophenanthridine

3）同义名：Hydroethidine，Hydroethidium,，PD-MY 003

4）分子式：C21H21N3

5）分子量：315.41

6）纯度：≥98%

7）Ex/Em：370/420nm（活细胞细胞质内荧光检测）；535/610nm（活细胞染色质荧光检测）

8）溶解性：溶于DMSO（10mg/ml），DMF，无水乙醇和氯仿（20 mg/ml）

9）化学结构式： 

保存与运输方法

保存：-20ºC避光干燥保存，至少1年有效。

运输：冰袋运输。

储存液制备

将冻干粉置于回温至少20min，加入适量高质量无水DMSO配制成5~10mM 储存液（比如，5mg DHE（Mw:315.41）加入1.58ml DMSO充分溶解即得到10mM储存液）。未用完的DMSO储存液请分装置于-20℃避光保存，最好置于惰性气体如氮气或氩气内。

使用方法

用生理缓冲液如PBS, HBSS, HEPES等稀释储存液到5~20µM工作液，然后加入等体积工作液到细胞内，37℃孵育5~60min加载探针（一般孵育30min即可得到均匀染色的细胞），随后简单清洗后，用流式细胞仪或其他适当荧光检测仪器进行检测。

可参考: A protocol for in vivo detection of reactive oxygen species

注意事项

1）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）本品对光敏感，保存和操作过程中注意避光。

3）本品氧化后产生一定乙锭类化合物，毒性较大，请注意防护。

4）为了您的安全和健康，请穿实验服并戴一次性手套操作。

相关产品

文献引用

1）Zhao AS et al. Hydrogen sulphide-releasing aspirin enhances cell capabilities of anti-oxidative lesions and anti-inflammation. Med Gas Res. 2019 Jul-Sep;9(3):145-152. PMID: 31552879

HUVECs and macrophages were respectively seeded on coverslips and cultured with samples for 24 hours and then treated with 400 μM H2O2 for 12 hours. After washing by PBS, cells were incubated with 10 μM ROS fluorescent probe-dihydroethidium (Maokangbio, Shanghai, China) at 37o C for 30 minutes away from light. Solution was removed for detection. Cells were observed and photographed at 488 nm using fluorescence microscope, with the fluorescent intensity measured by ImageJ software.

2）Wang DT et al. Curcumin ameliorates CKD-induced mitochondrial dysfunction and oxidative stress through inhibiting GSK-3β activity. The Journal of Nutritional Biochemistry Volume 83, September 2020, 108404

Dihydroethidium (DHE, #MX4812-25MG, MAOKANG Bioengineering Co., Shanghai, China) is an ethidium-based, redox-sensitive fluorescent probe shown to be oxidized by O2•− to form 2-hydroxyethidium. The assay was performed by using the previously protocol [39], and the fluorescence was measured at an excitation wavelength of 500–530 nm and an emission wavelength of 590–620 nm by using a fluorometer (Tecan, Switzerland).

[3] Zhou, Y., Yusufu, M., Zhang, T. et al. Silencing of miR-23a attenuates hydrogen peroxide (H2O2) induced oxidative damages in ARPE-19 cells by upregulating GLS1: an in vitro study. Cytotechnology 72, 873–884 (2020). https://doi.org/10.1007/s10616-020-00431-6 

24. 4% paraformaldehyde

Cell invasion was detected using transwell chamber (Corning, NY, USA). Briefly, cells were seeded in the upper chamber pre-coated with matrigel at a density of 1 × 105/chamber. The medium containing 10% FBS was added in the lower chamber. After 24 h of culturing, cells on the upper chamber were removed. Cells in the lower chamber were fixed with 4% paraformaldehyde (Shanghai Maokang Biotechnology Co., Ltd, Shanghai, China) for 15 min, and stained with 0.1% crystal violet for 30 min. Positive stained cells were counted under a microscope (Olympus) at five randomly selected fields, and the invasion rate was calculated.

The DHE reaction solution (MKbio, Shanghai, China) was added to the ARPE-19 cells according to the experimental instructions provided by the producer.

 — —Written/Edited by V. Shallan【版权归MKBio懋康所有】

上海懋康生物科技有限公司是一家涉足于生命科学和生物技术领域研究的试剂、仪器和实验室消耗品与实验服务工作，主要从事细胞生物学、植物学、分子生物学、免疫学、生物化学、蛋白组学。生物制药与诊断试剂研发生产等领域。 本公司秉承“以人为本，以诚为信、合同守信”的经营理念。坚持"品质保障"的原则为广大客户提供优质产品。