细胞内活性氧ROS检测方案─YEASEN攻略大放送

活性氧(Reactive Oxygen Species, ROS)是氧代谢产生的化学活性含氧分子，包括过氧化物，超氧化物，羟基自由基，单线态氧和α-氧。在生物学背景下，ROS的形成在细胞信号传导和体内平衡中具有重要作用。然而，在环境压力（例如，紫外线或热暴露）期间，ROS水平会急剧增加，导致氧化应激，从而会造成细胞蛋白质、脂质和DNA受损，并与心血管疾病、癌症、糖尿病、炎症和衰老等有关。

YEASEN生物50101ES Reactive Oxygen Species Assay Kit活性氧(ROS)检测试剂盒是一种基于荧光染料DCFH-DA (2,7-Dichlorodi -hydrofluorescein diacetate)的荧光强度变化，定量检测细胞内活性氧水平的最常用方法。DCFH-DA本身没有荧光，可以自由穿过细胞膜。进入细胞内后被细胞内的酯酶水解生成DCFH，而DCFH不会通透细胞膜，因此探针很容易被积聚在细胞内。细胞内的活性氧能够氧化无荧光的DCFH生成有荧光的DCF (λex = 490 nm/λem=525 nm)，绿色荧光强度与活性氧的水平成正比。

图1 YEASEN 50101ES Reactive Oxygen Species Assay Kit活性氧(ROS)检测试剂盒

实验过程

1. 原位装载探针

1）细胞准备：检测前一天进行细胞铺板，确保检测时细胞汇合度达到50~70%。

【注】：必须保证细胞状态健康，且检测时不会过度生长。

2）药物诱导：去除细胞培养液，加入适量经合适的缓冲液或无血清培养基稀释到工作浓度的药物，于37℃细胞培养箱内孵育，具体诱导时间根据药物特性以及细胞类型来决定。

（可选）阳性对照：先用无血清培养基等稀释阳性对照(Rosup, 100 mM)到常用工作浓度100 μM，加入细胞，一般37℃避光孵育0.5 ̴ 4 h可显著看到ROS水平提高，但依细胞类型会有比较明显差异。如，HeLa细胞孵育30 min；MRC5人胚胎成纤维细胞1.5 h。

3）探针准备：按照1:1000用无血清培养液稀释DCFH-DA，使其终浓度为10 μM。

4）探针装载：吸除诱导用药物，加入适当体积稀释好的DCFH-DA工作液。

注：对于贴壁细胞，工作液以能充分盖住细胞为宜。例如；对于6孔板不少于1 mL，对于96孔板不少于100 μL，37℃细胞培养箱内避光孵育30 min。对于悬浮细胞，单管内细胞数目不少于104，不可多于106，密度为1.0×106~2.0×107。每隔3-5 min颠倒混匀一下，使探针和细胞充分接触。

5）细胞清洗：用无血清培养液洗涤细胞1~2次，充分去除未进入细胞内的DCFH-DA。

2. 荧光检测及参数设置

用荧光分光光度计、荧光酶标仪或流式细胞仪检测，也可以用激光共聚焦显微镜直接观察。使用488 nm激发波长，525 nm发射波长，实时或逐时间点检测刺激前后荧光的强弱。DCF的荧光光谱和FITC非常相似，可以用FITC的参数设置检测DCF。DCF的激发光谱和发射光谱图如下：

图2 DCF的激发光谱和发射光谱图

本产品相关结果

本试剂盒主要用于检测动物活细胞内的ROS水平，一般不能检测组织切片，对于活体组织内的检测没有验证过，可以将活体组织分散成单细胞后再进行检测。

图3 4T1细胞辐射后使用YEASEN 50101ES试剂盒染色检测ROS的荧光图( PMID: 31351244 IF: 11.591)^[1]

图4 MDA-MB-231细胞在PP3-Se纳米颗粒和光(660 nm laser, 0.75 W/cm2, 4 min)处理后使用YEASEN 50101ES试剂盒染色检测ROS的流式图(PMID: 31569018 IF: 11.591)^[2]

FAQ

已发表文章（部分）

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