CellROX Deep Red Reagent, for oxidative stress detection

氧化應激檢測用探針（深紅(hóng)色）

溫馨提示：

見(jiàn)我司（懋康生(shēng)物(wù)）整理的活性氧ROS熒光(guāng)探針專題。

見(jiàn)我司（懋康生(shēng)物(wù)）整理的CellROX Reagent, for oxidative stress detection氧化應激檢測探針産品專題。

訂購信息：

背景介紹：

氧化應激（Oxidative stress）的産生(shēng)，起因于反應氧物(wù)質（ROS）生(shēng)成與細胞清除ROS之間的不平衡。ROS在幾種疾病的發展中發揮重要作用，包括炎症、動脈粥樣硬化、衰老和衰老相(xiàng)關的退行性疾病。

基本介紹：

CellROX Deep Red Reagent是一(yī)種檢測活細胞氧化應激的新型熒光(guāng)探針。此細胞膜滲透性的染料在還(hái)原态時無熒光(guāng)或呈微弱熒光(guāng)，一(yī)旦被活性氧物(wù)質（ROS）氧化後發明亮穩定的熒光(guāng)，最大吸收/發射波長(cháng)約~ 644/ 665nm（見(jiàn)圖I.CellROX Deep Red氧化後的熒光(guāng)光(guāng)譜圖）。其熒光(guāng)信号主要定位在細胞質。

CellROX Deep Red Reagent的深紅(hóng)色熒光(guāng)信号與其他活細胞染料和GFP兼容，使其适用于多(duō)重熒光(guāng)試驗來測定各種細胞現象，包括與細胞毒性和細胞死亡的參數。不同于其它的ROS探針，比如H2DCFDA（MX4802-50MG），CellROX Deep Red Reagent的熒光(guāng)信号經醛類固定後也能(néng)保留，使其相(xiàng)對于傳統的ROS探針來說在應用和操作流程更加靈活。CellROX Deep Red Reagent與各種檢測平台兼容，比如傳統熒光(guāng)顯微鏡、高(gāo)内涵篩選儀器(qì)（HCS）、流式細胞儀、熒光(guāng)酶标儀或高(gāo)通(tōng)量篩選（HTS）。還(hái)與各種台式儀器(qì)兼容，比如Attune™儀器(qì)。

産品參數

1. 外觀：無色溶液（Colorless solution）

2. 溶劑：DMSO

3. 激發/發射光(guāng)譜範圍：644/665nm（可見(jiàn)光(guāng)/可見(jiàn)光(guāng)）

4. 亞細胞定位：細胞質

5. 熒光(guāng)顔色：遠(yuǎn)紅(hóng)（Far-red）

6. 保存方法：≤-20°C避光(guāng)幹燥

染色示例

Fig1. BPAE detection of oxidative stress using MitoTracker® Green (Cat. No. M7514) and CellROX® Deep Red Reagent (Cat. No. C10422) BPAE cells were plated in a 96 well plate, treated with 100 µM menadione for 1 hr and then stained with 20 nM MitoTracker® Green, 5 µM Far Red ROS Sensor and Hoechst 33342 for 30 mins in complete medium. Cells were then washed 3X with PBS and imaged on a Thermo Fisher Cellomics ArrayScan® VTI. The control sample has no signal while in menadione treated cells, there is a robust increase in the signal as a result of oxidative stress caused by menadione. Both MitoTracker® Green and Far Red ROS Sensor stained well showing that they work together in a multiplex assay.

Fig2. A549 spheroid mounted with Prolong Glass and imaged with EVOS FL Auto 2. A549 spheroid grown in Nunclon Sphera microplates (Cat. No. 174925) at 1000 cells/well. Live cells were labeled with NucBlue Live ReadyProbes Reagent (Cat. No. R37605), MitoTracker Orange (Cat. No. M7510), and CellROX Deep Red Reagent for oxidative stress detection (Cat. No. C10422) for 1 hour at 37°C. Cells were then fixed with 4% PFA, transferred to a #1.5 coverslip, and mounted in ProLong Glass. Sample was allowed to cure overnight exposed to air, and the coverslip was mounted to the slide using 100% glycerol. Image acquired with EVOS FL Auto 2 (AMAFD2000) using an Olympus 10X air objective (AMEP4753).

常見(jiàn)問題

1）I am trying to label my cells with a reactive oxygen species (ROS) indicator dye, but I am not seeing a significant difference in signal. What could be happening?

First, make sure you have both a negative (untreated) and positive (ROS-induced) sample to compare. A good positive control can be the use of 100 μM menadione for one hour or 50 μM nefazodone for 24 hours. H2O2 can also be used, though it does not work well for CellROX™ dyes. Some dyes, such as H2DCFDA, require esterase cleavage, so don't incubate in the presence of serum (which contains esterases that can prematurely cleave the dye). If your positive control does not show significant change compared to the negative control, try increasing the concentration and label time for the dye. Our manuals give starting recommendations. Be sure to image your live cells as soon as possible. Only two dyes (CellROX™ Green and CellROX™ Deep Red) are retained with formaldehyde fixation. Finally, make sure you are using filters and instrument settings to match the excitation and emission spectra of the dye.

2）I need a formaldehyde-fixable reactive oxygen species detection assay. Is H2DCFDA fixable?

H2DCFDA and similar derivatives are not fixable. The same goes for dihydroethidium and dihydrorhodamine. However, CellROX™ Deep Red and CellROX™ Green are retained for a limited time upon fixation with formaldehyde. CellROX™ Green may be retained upon subsequent Triton™ X-100 permeabilization. Avoid the use of any acetone or alcohol-based fixatives or fixatives that include alcohol, such as formalin.

3) What dyes can I use to detect reactive oxygen species (ROS) in my bacteria?

Many dyes that are used on mammalian cells have also been shown to be useful in bacterial cells. For example, CellROX Deep Red Reagent has been shown to work in B. subtilis (see Reference: http://www-brs.ub.ruhr-uni-bochum.de/netahtml/HSS/Diss/RaatschenNadja/diss.pdf). If you are interested in a particular dye, but are not sure if it will work on your bacteria, literature searches are the best way to check to see if it has been tested. If not, then it may be worth testing yourself.

注意事(shì)項

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。

 — —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。