ROSGreen^TM H₂O₂ Probe 過氧化氫熒光(guāng)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(guāng)素; Mitosox Red;

同義名：

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

産品信息

産品描述

過氧化氫（Hydrogen peroxide, H2O2）是一(yī)種反映氧代謝副産物(wù)，用于許多(duō)氧化應激相(xiàng)關态的關鍵調節劑，參與人體大量的生(shēng)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(shēng)成、積累、運輸和功能(néng)的分子機(jī)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(néng)得到(dào)的H2O2反應性探針的缺陷包括1）來自(zì)其他ROS比如過氧亞硝基陰離子（ONOO-）、過氧化基（ROO•）、超氧化物(wù)（•O2–）的背景熒光(guāng)幹擾；2）對外源活化酶的需求；3）水(shuǐ)缺乏水(shuǐ)溶性或不兼容于水(shuǐ)溶體系；4）以及紫外區域的激發光(guāng)譜特征等。

本ROSGreenTMH2O2Probe正是克服以上(shàng)缺陷開(kāi)發的一(yī)款過氧化氫（H2O2）選擇性探針。探針本身無熒光(guāng)，可見(jiàn)光(guāng)區域内無吸收峰。一(yī)旦接觸H2O2，瞬間激發熒光(guāng)增加并伴随可見(jiàn)波長(cháng)吸收帶的生(shēng)成（Ex/Em= 490/514nm）。因其二元的吸收/發射熒光(guāng)反應，此探針具有較大的動力學範圍。與相(xiàng)似的ROS比如O2–，NO，或OCl-相(xiàng)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(guāng)幹燥保存，至少1年(nián)有效。

運輸：冰袋運輸。

注意事(shì)項

1）本品的DMSO儲存液需分裝成單次用量後，-20ºC避光(guāng)保存，避免反複凍融。所有工(gōng)作液現配現用。

2）整個(gè)操作過程中注意避光(guāng)。

3）為(wèi)了您的安全和健康，請穿實驗服并戴一(yī)次性手套操作。

使用方法

1、儲存液制備

将低(dī)溫保存的産品從(cóng)冰箱取出，置于室溫回溫至少20min。低(dī)速離心3-5min。之後往瓶内加入适量無水(shuǐ)DMSO充分溶解配成2~5mM儲存液（比如，1mg ROSGreenTMH2O2Probe（Mw: ~ 600）加入333μl DMSO，充分溶解混勻後即得到(dào)5mM儲存液）。按照(zhào)單次用量分裝凍存，避免反複凍融，至少3個(gè)月(yuè)穩定。

2、工(gōng)作液準備

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

2.1 對于溶液H2O2測定，按照(zhào)2~20μM工(gōng)作液濃度制備2×工(gōng)作液，用20mM Hepes緩沖液或其他緩沖液（pH 7.0）稀釋儲存液。工(gōng)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(zhào)2~20μM工(gōng)作液濃度制備1×工(gōng)作液，用含20mM Hepes緩沖液的HBSS，pH 7.0 或PBS稀釋儲存液。工(gōng)作液現配現用。

3、 上(shàng)清液H2O2測定

【以下(xià)為(wèi)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。】

3.1 于96孔闆，加50μl 2×ROSGreenTMH2O2Probe工(gōng)作液到(dào)每個(gè)孔（H2O2标準、空白(bái)對照(zhào)、待測樣本）内使總的測定體積為(wèi)100μl/孔。【注意：對于384孔闆，加25μl樣品和25μl 2×ROSGreenTMH2O2Probe工(gōng)作液，使得測定體積為(wèi)50μl/孔。】

3.2 室溫避光(guāng)孵育反應體系15~60min。

3.3 熒光(guāng)酶标闆内監測用熒光(guāng)增強，Ex/Em=490/525nm。

3.4 空白(bái)孔（隻含有反應緩沖液）的熒光(guāng)用作對照(zhào)，其他孔的熒光(guāng)值需減掉空白(bái)對照(zhào)孔熒光(guāng)。

4、活細胞H2O2測定

【ROSGreenTMH2O2Probe能(néng)主動擴散進入活細胞，且能(néng)反映胞内H2O2微摩爾濃度變化。以下(xià)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。】

4.1 按照(zhào)步驟2.2配制1×ROSGreenTMH2O2Probe工(gōng)作液。工(gōng)作液現配現用。

4.2 按照(zhào)實驗要求處理細胞。

4.3 用1×ROSGreenTMH2O2Probe工(gōng)作液避光(guāng)孵育細胞5~60min或适合自(zì)身需求的時間。用PBS清洗細胞2次。

4.3 用熒光(guāng)酶标闆（底部讀(dú)數模式）監測熒光(guāng)增強，Ex/Em=490/525nm。或者用熒光(guāng)顯微鏡的FITC通(tōng)道成像檢測熒光(guāng)增強。

染色示例

圖1. Costar 黑(hēi)色96孔培養闆内CHO-K1活細胞用ROSGreenTMH2O2Probe的染色圖。圖A：對照(zhào)細胞；圖B：用H2O2（100μM）處理5min後的細胞；

相(xiàng)關産品

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). 

 — —Written/Edited by V. Shallan【版權歸MKBio懋康所有】

上海夢澤生物科技有限公司是一(yī)家涉足于生(shēng)命科學和生(shēng)物(wù)技(jì)術(shù)領域研究的試劑、儀器(qì)和實驗室消耗品與實驗服務工(gōng)作，主要從(cóng)事(shì)細胞生(shēng)物(wù)學、植物(wù)學、分子生(shēng)物(wù)學、免疫學、生(shēng)物(wù)化學、蛋白(bái)組學。生(shēng)物(wù)制藥與診斷試劑研發生(shēng)産等領域。 本公司秉承“以人為(wèi)本，以誠為(wèi)信、合同守信”的經營理念。堅持"品質保障"的原則為(wèi)廣大客戶提供優質産品。