ROSGreen^TM H₂O₂ Probe  过氧化氢荧光探针

搜索关键词：

Hydrogen Peroxide 过氧化氢；H2DCFDA (DCFH-DA, DCFH)；BES-H2O2-Ac；APF 氨基苯基荧光素; Mitosox Red;

同义名：

ROSGreen^TM Hydrogen peroxide Probe；ROSGreen^TM Hydrogen peroxide sensor；ROSGreen^TM H2O2sensor；

产品信息

产品描述

过氧化氢（Hydrogen peroxide, H2O2）是一种反映氧代谢副产物，用于许多氧化应激相关态的关键调节剂，参与人体大量的生理和病理活动，比如哮喘、动脉粥样硬化、糖尿病血管病变、神经性衰退疾病和唐氏综合征等，但目前对H2O2生成、积累、运输和功能的分子机制仍了解很少，最大的限制在于缺乏灵敏且特异性H2O2探针，用于活细胞研究。目前能得到的H2O2反应性探针的缺陷包括1）来自其他ROS比如过氧亚硝基阴离子（ONOO-）、过氧化基（ROO•）、超氧化物（•O2–）的背景荧光干扰；2）对外源活化酶的需求；3）水缺乏水溶性或不兼容于水溶体系；4）以及紫外区域的激发光谱特征等。

本ROSGreenTMH2O2Probe正是克服以上缺陷开发的一款过氧化氢（H2O2）选择性探针。探针本身无荧光，可见光区域内无吸收峰。一旦接触H2O2，瞬间激发荧光增加并伴随可见波长吸收带的生成（Ex/Em= 490/514nm）。因其二元的吸收/发射荧光反应，此探针具有较大的动力学范围。与相似的ROS比如O2–，NO，或OCl-相比，ROSGreenTMH2O2Probe对H2O2呈现出＞100倍（100-fold）的选择性。

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。

注意事项

1）本品的DMSO储存液需分装成单次用量后，-20ºC避光保存，避免反复冻融。所有工作液现配现用。

2）整个操作过程中注意避光。

3）为了您的安全和健康，请穿实验服并戴一次性手套操作。

使用方法

1、储存液制备

将低温保存的产品从冰箱取出，置于室温回温至少20min。低速离心3-5min。之后往瓶内加入适量无水DMSO充分溶解配成2~5mM储存液（比如，1mg ROSGreenTMH2O2Probe（Mw: ~ 600）加入333μl DMSO，充分溶解混匀后即得到5mM储存液）。按照单次用量分装冻存，避免反复冻融，至少3个月稳定。

2、工作液准备

正式实验前，或取粉末按照【储存液制备】用DMSO溶解，或于室温融化一管DMSO储存液。

2.1 对于溶液H2O2测定，按照2~20μM工作液浓度制备2×工作液，用20mM Hepes缓冲液或其他缓冲液（pH 7.0）稀释储存液。工作液现配现用。建议使用5μM终浓度的ROSGreenTMH2O2Probe，测定溶液体系中H2O2浓度。

2.2 对于细胞H2O2测定，按照2~20μM工作液浓度制备1×工作液，用含20mM Hepes缓冲液的HBSS，pH 7.0 或PBS稀释储存液。工作液现配现用。

3、 上清液H2O2测定

【以下为溶液H2O2测定的推荐步骤，仅作参考，请根据具体需求做优化调整。】

3.1 于96孔板，加50μl 2×ROSGreenTMH2O2Probe工作液到每个孔（H2O2标准、空白对照、待测样本）内使总的测定体积为100μl/孔。【注意：对于384孔板，加25μl样品和25μl 2×ROSGreenTMH2O2Probe工作液，使得测定体积为50μl/孔。】

3.2 室温避光孵育反应体系15~60min。

3.3 荧光酶标板内监测用荧光增强，Ex/Em=490/525nm。

3.4 空白孔（只含有反应缓冲液）的荧光用作对照，其他孔的荧光值需减掉空白对照孔荧光。

4、活细胞H2O2测定

【ROSGreenTMH2O2Probe能主动扩散进入活细胞，且能反映胞内H2O2微摩尔浓度变化。以下是推荐的活细胞H2O2显微成像检测步骤，仅作参考，可根据具体需求优化调整。】

4.1 按照步骤2.2配制1×ROSGreenTMH2O2Probe工作液。工作液现配现用。

4.2 按照实验要求处理细胞。

4.3 用1×ROSGreenTMH2O2Probe工作液避光孵育细胞5~60min或适合自身需求的时间。用PBS清洗细胞2次。

4.3 用荧光酶标板（底部读数模式）监测荧光增强，Ex/Em=490/525nm。或者用荧光显微镜的FITC通道成像检测荧光增强。

染色示例

图1. Costar 黑色96孔培养板内CHO-K1活细胞用ROSGreenTMH2O2Probe的染色图。图A：对照细胞；图B：用H2O2（100μM）处理5min后的细胞；

相关产品

引用文献

[1]Ma W, Chen X, Fu L, et al. Ultra-efficient Antibacterial System Based on Photodynamic Therapy and CO Gas Therapy for Synergistic Antibacterial and Ablation Biofilms. ACS Appl Mater Interfaces. 2020;12(20):22479-22491. doi:10.1021/acsami.0c01967

Variation of ROS and H2O2 level in E. coli after CO release was respectively measured by

DCFH-DA (10 μΜ) and ROSGreenTM H2O2 Probe (5 μM) using the parallel method. The λEx and λEm for DCFH-DA were 505 and 535 nm, respectively, while that for ROSGreenTM H2O2 Probe were 490 and 525 nm, respectively. DCFH-DA and ROSGreenTM H2O2 Probe were purchased from Maokang Co., Ltd. (Shanghai,China).

[2]https://pubmed.ncbi.nlm.nih.gov/31815453/

Luo X, Wang R, Lv C, Chen G, You J, Yu F. Detection of Selenocysteine with a Ratiometric near-Infrared Fluorescent Probe in Cells and in Mice Thyroid Diseases Model. Anal Chem. 2020;92(1):1589-1597. doi:10.1021/acs.analchem.9b04860

Besides, both of our probes Mito-Cy-Sec and commercial ROSGreen H2O2 are employed to examine the interrelationship between H2O2 and Sec in cells and in mice models. The results demonstrate that the relevant-levels between H2O2 and Sec are exactly negative correlation.

[3] Guangzhen Yang, Mengni Fan, Jingwu Zhu, Chen Ling, Lihuang Wu, Xin Zhang, Ming Zhang, Jiayi Li, Qingqiang Yao, Zhongwei Gu, Xiaojun Cai, A multifunctional anti-inflammatory drug that can specifically target activated macrophages, massively deplete intracellular H2O2, and produce large amounts CO for a highly efficient treatment of osteoarthritis, Biomaterials, Volume 255, 2020, 120155, ISSN 0142-9612,

https://doi.org/10.1016/j.biomaterials.2020.120155.

ROSGreen™ H 2 O 2 Probe was purchased from (MaokangBio, China).

[4] Taotao Zhu, Ziwen Li, Xueli An, Yan Long, Xiaofeng Xue, Ke Xie, Biao Ma, Danfeng Zhang, Yijian Guan, Canfang Niu, Zhenying Dong, Quancan Hou, Lina Zhao, Suowei Wu, Jinping Li, Weiwei Jin, Xiangyuan Wan,

Normal Structure and Function of Endothecium Chloroplasts Maintained by ZmMs33-Mediated Lipid Biosynthesis in Tapetal Cells Are Critical for Anther Development in Maize, Molecular Plant, Volume 13, Issue 11,2020,Pages 1624-1643,ISSN 1674-2052, https://doi.org/10.1016/j.molp.2020.09.013.

For ROS and H2O2 measurement, anthers were stained with the fluorescent dyes H2DCF-DA (Sigma-Aldrich) and ROSGreen (a special H2O2 Probe) (MKBio, China), respectively, as described by Huang et al. (2013) and Xie et al. (2014). The relative fluorescence intensity of whole anthers was quantified using ImageJ software.

[5] Siyu Wang, Yue Zhou, Xiaoyang Liang, Min Xu, Nan Li, Kang Zhao,Platinum-cerium bimetallic nano-raspberry for atherosclerosis treatment via synergistic foam cell inhibition and P2Y12 targeted antiplatelet aggregation, Chemical Engineering Journal,Volume 430, Part 1,2022,132859,ISSN 1385-8947,

https://doi.org/10.1016/j.cej.2021.132859.

[6] He Y, Jin X, Guo S, Zhao H, Liu Y, Ju H. Conjugated Polymer-Ferrocence Nanoparticle as an NIR-II Light Powered Nanoamplifier to Enhance Chemodynamic Therapy. ACS Appl Mater Interfaces. 2021 Jul 14;13(27):31452-31461. doi: 10.1021/acsami.1c06613. Epub 2021 Jul 1. PMID: 34197086.

[7] Li H, Zhang Y, Liang L, Song J, Wei Z, Yang S, Ma Y, Chen WR, Lu C, Wen L. Doxorubicin-Loaded Metal-Organic Framework Nanoparticles as Acid-Activatable Hydroxyl Radical Nanogenerators for Enhanced Chemo/Chemodynamic Synergistic Therapy. Materials (Basel). 2022 Jan 30;15(3):1096. doi: 10.3390/ma15031096. PMID: 35161041; PMCID: PMC8838206.

ROSGreen H 2 O 2 probes were purchased from Shanghai Maokang Bio-technology, China.

[8] Zhang YY, Ren H, Yan QL, Li YL, Liu Q, Yao GD, Song SJ. SCP-7, a germacrane-type sesquiterpene lactone derivative, induces ROS-mediated apoptosis in NSCLC cells in vitro and in vivo. Eur J Pharmacol. 2022 Jun 15;925:174989. doi: 10.1016/j.ejphar.2022.174989. Epub 2022 Apr 28. PMID: 35490722.

 HPF (Hydroxyphenyl fluorescein) and ROS GreenTM H2 O 2 Probe was purchased from Shanghai Maokang Bio. Co. (Shanghai, China). 

[9] Zhang H, Qu H, He Q, et al. Thrombus-targeted nanoparticles for thrombin-triggered thrombolysis and local inflammatory microenvironment regulation. Journal of Controlled Release : Official Journal of the Controlled Release Society. 2021 Nov;339:195-207. DOI: 10.1016/j.jconrel.2021.06.043. PMID: 34214595.

 ROSGreen™ H 2 O 2 Probe was purchased from Maokang Co., Ltd. (Shanghai, China).

[10] Junpeng Chen et al. An all-in-one CO gas therapy-based hydrogel dressing with sustained insulin release, anti-oxidative stress, antibacterial, and anti-inflammatory capabilities for infected diabetic wounds. Acta Biomaterialia. Volume 146, 1 July 2022, Pages 49-65

 ROSGreen™ H 2 O 2 Probe was obtained from (MaokangBio, China). 

 — —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。