ROSGreen^TM H₂O₂ Probe  過氧化氫熒光探針

搜索關鍵詞：

Hydrogen Peroxide 過氧化氫；H2DCFDA (DCFH-DA, DCFH)；BES-H2O2-Ac；APF 氨基苯基熒光素; Mitosox Red;

同義名：

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

產品信息

產品描述

過氧化氫（Hydrogen peroxide, H2O2）是一種反映氧代謝副產物，用于許多氧化應激相關態的關鍵調節劑，參與人體大量的生理和病理活動，比如哮喘、動脈粥樣硬化、糖尿病血管病變、神經性衰退疾病和唐氏綜合征等，但目前對H2O2生成、積累、運輸和功能的分子機制仍了解很少，最大的限制在于缺乏靈敏且特異性H2O2探針，用于活細胞研究。目前能得到的H2O2反應性探針的缺陷包括1）來自其他ROS比如過氧亞硝基陰離子（ONOO-）、過氧化基（ROO?）、超氧化物（?O2–）的背景熒光干擾；2）對外源活化酶的需求；3）水缺乏水溶性或不兼容于水溶體系；4）以及紫外區域的激發光譜特征等。

本ROSGreenTMH2O2Probe正是克服以上缺陷開發的一款過氧化氫（H2O2）選擇性探針。探針本身無熒光，可見光區域內無吸收峰。一旦接觸H2O2，瞬間激發熒光增加并伴隨可見波長吸收帶的生成（Ex/Em= 490/514nm）。因其二元的吸收/發射熒光反應，此探針具有較大的動力學范圍。與相似的ROS比如O2–，NO，或OCl-相比，ROSGreenTMH2O2Probe對H2O2呈現出＞100倍（100-fold）的選擇性。

保存與運輸方法

保存：-20oC避光干燥保存，至少1年有效。

運輸：冰袋運輸。

注意事項

1）本品的DMSO儲存液需分裝成單次用量后，-20oC避光保存，避免反復凍融。所有工作液現配現用。

2）整個操作過程中注意避光。

3）為了您的安全和健康，請穿實驗服并戴一次性手套操作。

使用方法

1、儲存液制備

將低溫保存的產品從冰箱取出，置于室溫回溫至少20min。低速離心3-5min。之后往瓶內加入適量無水DMSO充分溶解配成2~5mM儲存液（比如，1mg ROSGreenTMH2O2Probe（Mw: ~ 600）加入333μl DMSO，充分溶解混勻后即得到5mM儲存液）。按照單次用量分裝凍存，避免反復凍融，至少3個月穩定。

2、工作液準備

正式實驗前，或取粉末按照【儲存液制備】用DMSO溶解，或于室溫融化一管DMSO儲存液。

2.1 對于溶液H2O2測定，按照2~20μM工作液濃度制備2×工作液，用20mM Hepes緩沖液或其他緩沖液（pH 7.0）稀釋儲存液。工作液現配現用。建議使用5μM終濃度的ROSGreenTMH2O2Probe，測定溶液體系中H2O2濃度。

2.2 對于細胞H2O2測定，按照2~20μM工作液濃度制備1×工作液，用含20mM Hepes緩沖液的HBSS，pH 7.0 或PBS稀釋儲存液。工作液現配現用。

3、 上清液H2O2測定

【以下為溶液H2O2測定的推薦步驟，僅作參考，請根據具體需求做優化調整?！?/span>

3.1 于96孔板，加50μl 2×ROSGreenTMH2O2Probe工作液到每個孔（H2O2標準、空白對照、待測樣本）內使總的測定體積為100μl/孔?！咀⒁猓簩τ?84孔板，加25μl樣品和25μl 2×ROSGreenTMH2O2Probe工作液，使得測定體積為50μl/孔?！?/span>

3.2 室溫避光孵育反應體系15~60min。

3.3 熒光酶標板內監測用熒光增強，Ex/Em=490/525nm。

3.4 空白孔（只含有反應緩沖液）的熒光用作對照，其他孔的熒光值需減掉空白對照孔熒光。

4、活細胞H2O2測定

【ROSGreenTMH2O2Probe能主動擴散進入活細胞，且能反映胞內H2O2微摩爾濃度變化。以下是推薦的活細胞H2O2顯微成像檢測步驟，僅作參考，可根據具體需求優化調整?！?/span>

4.1 按照步驟2.2配制1×ROSGreenTMH2O2Probe工作液。工作液現配現用。

4.2 按照實驗要求處理細胞。

4.3 用1×ROSGreenTMH2O2Probe工作液避光孵育細胞5~60min或適合自身需求的時間。用PBS清洗細胞2次。

4.3 用熒光酶標板（底部讀數模式）監測熒光增強，Ex/Em=490/525nm?；蛘哂脽晒怙@微鏡的FITC通道成像檢測熒光增強。

染色示例

圖1. Costar 黑色96孔培養板內CHO-K1活細胞用ROSGreenTMH2O2Probe的染色圖。圖A：對照細胞；圖B：用H2O2（100μM）處理5min后的細胞；

相關產品

引用文獻

[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【版權歸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學和生物技術領域研究的試劑、儀器和實驗室消耗品與實驗服務工作，主要從事細胞生物學、植物學、分子生物學、免疫學、生物化學、蛋白組學。生物制藥與診斷試劑研發生產等領域。 本公司秉承“以人為本，以誠為信、合同守信”的經營理念。堅持"品質保障"的原則為廣大客戶提供優質產品。