細(xì)胞內(nèi)活性氧ROS檢測(cè)方案─YEASEN攻略大放送
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活性氧(Reactive Oxygen Species, ROS)是氧代謝產(chǎn)生的化學(xué)活性含氧分子，包括過(guò)氧化物，超氧化物，羥基自由基，單線(xiàn)態(tài)氧和α-氧。在生物學(xué)背景下，ROS的形成在細(xì)胞信號(hào)傳導(dǎo)和體內(nèi)平衡中具有重要作用。然而，在環(huán)境壓力（例如，紫外線(xiàn)或熱暴露）期間，ROS水平會(huì)急劇增加，導(dǎo)致氧化應(yīng)激，從而會(huì)造成細(xì)胞蛋白質(zhì)、脂質(zhì)和DNA受損，并與心血管疾病、癌癥、糖尿病、炎癥和衰老等有關(guān)。
YEASEN生物50101ES Reactive Oxygen Species Assay Kit活性氧(ROS)檢測(cè)試劑盒是一種基于熒光染料DCFH-DA (2,7-Dichlorodi -hydrofluorescein diacetate)的熒光強(qiáng)度變化，定量檢測(cè)細(xì)胞內(nèi)活性氧水平的最常用方法。DCFH-DA本身沒(méi)有熒光，可以自由穿過(guò)細(xì)胞膜。進(jìn)入細(xì)胞內(nèi)后被細(xì)胞內(nèi)的酯酶水解生成DCFH，而DCFH不會(huì)通透細(xì)胞膜，因此探針很容易被積聚在細(xì)胞內(nèi)。細(xì)胞內(nèi)的活性氧能夠氧化無(wú)熒光的DCFH生成有熒光的DCF (λex = 490 nm/λem=525 nm)，綠色熒光強(qiáng)度與活性氧的水平成正比。
圖1 YEASEN 50101ES Reactive Oxygen Species Assay Kit活性氧(ROS)檢測(cè)試劑盒
實(shí)驗(yàn)過(guò)程
1. 原位裝載探針
1）細(xì)胞準(zhǔn)備：檢測(cè)前一天進(jìn)行細(xì)胞鋪板，確保檢測(cè)時(shí)細(xì)胞匯合度達(dá)到50~70%。
【注】：必須保證細(xì)胞狀態(tài)健康，且檢測(cè)時(shí)不會(huì)過(guò)度生長(zhǎng)。
2）藥物誘導(dǎo)：去除細(xì)胞培養(yǎng)液，加入適量經(jīng)合適的緩沖液或無(wú)血清培養(yǎng)基稀釋到工作濃度的藥物，于37℃細(xì)胞培養(yǎng)箱內(nèi)孵育，具體誘導(dǎo)時(shí)間根據(jù)藥物特性以及細(xì)胞類(lèi)型來(lái)決定。
（可選）陽(yáng)性對(duì)照：先用無(wú)血清培養(yǎng)基等稀釋陽(yáng)性對(duì)照(Rosup, 100 mM)到常用工作濃度100 μM，加入細(xì)胞，一般37℃避光孵育0.5 ? 4 h可顯著看到ROS水平提高，但依細(xì)胞類(lèi)型會(huì)有比較明顯差異。如，HeLa細(xì)胞孵育30 min；MRC5人胚胎成纖維細(xì)胞1.5 h。
3）探針準(zhǔn)備：按照1:1000用無(wú)血清培養(yǎng)液稀釋DCFH-DA，使其終濃度為10 μM。
4）探針裝載：吸除誘導(dǎo)用藥物，加入適當(dāng)體積稀釋好的DCFH-DA工作液。
注：對(duì)于貼壁細(xì)胞，工作液以能充分蓋住細(xì)胞為宜。例如；對(duì)于6孔板不少于1 mL，對(duì)于96孔板不少于100 μL，37℃細(xì)胞培養(yǎng)箱內(nèi)避光孵育30 min。對(duì)于懸浮細(xì)胞，單管內(nèi)細(xì)胞數(shù)目不少于104，不可多于106，密度為1.0×106~2.0×107。每隔3-5 min顛倒混勻一下，使探針和細(xì)胞充分接觸。
5）細(xì)胞清洗：用無(wú)血清培養(yǎng)液洗滌細(xì)胞1~2次，充分去除未進(jìn)入細(xì)胞內(nèi)的DCFH-DA。
2. 熒光檢測(cè)及參數(shù)設(shè)置
用熒光分光光度計(jì)、熒光酶標(biāo)儀或流式細(xì)胞儀檢測(cè)，也可以用激光共聚焦顯微鏡直接觀察。使用488 nm激發(fā)波長(zhǎng)，525 nm發(fā)射波長(zhǎng)，實(shí)時(shí)或逐時(shí)間點(diǎn)檢測(cè)刺激前后熒光的強(qiáng)弱。DCF的熒光光譜和FITC非常相似，可以用FITC的參數(shù)設(shè)置檢測(cè)DCF。DCF的激發(fā)光譜和發(fā)射光譜圖如下：
圖2 DCF的激發(fā)光譜和發(fā)射光譜圖
本產(chǎn)品相關(guān)結(jié)果
本試劑盒主要用于檢測(cè)動(dòng)物活細(xì)胞內(nèi)的ROS水平，一般不能檢測(cè)組織切片，對(duì)于活體組織內(nèi)的檢測(cè)沒(méi)有驗(yàn)證過(guò)，可以將活體組織分散成單細(xì)胞后再進(jìn)行檢測(cè)。
圖3 4T1細(xì)胞輻射后使用YEASEN 50101ES試劑盒染色檢測(cè)ROS的熒光圖( PMID: 31351244 IF: 11.591)[1]
圖4 MDA-MB-231細(xì)胞在PP3-Se納米顆粒和光(660 nm laser, 0.75 W/cm2, 4 min)處理后使用YEASEN 50101ES試劑盒染色檢測(cè)ROS的流式圖(PMID: 31569018 IF: 11.591)[2]
FAQ
已發(fā)表文章（部分）
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