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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。

image1.png

 

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:

image2.png 

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。

 image3.png

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]

image4.png

 

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

Q

A

1.使用范围

一般用于哺乳动物细胞,对于植物或是细菌可以在制备原生质体后进行检测使用,该试剂盒不能检测体内的ROS。

2.如何避免过高的荧光背景

探针孵育后,一定要洗净残余的未进入细胞内的探针

3.可以检测正常细胞中的ROS含量吗

正常细胞中活性氧含量很低,检测效果不会很好。

4.阴性和阳性荧光值一样

加入的探针浓度过大导致的,建议适当降低探针浓度:5-7.5 μM,同时降低孵育时间:15-20 min。

5.同一支探针,未分装,前5次效果很好, 单这次没有染上

1.细胞状态不好,导致染色效率低;2.阳性药物诱导时间过短,一般 37℃避光孵育 30 min-4 h 可显著看到活性氧水平提高;3.探针反复冻融4次以上,染色效率降低,荧光信号不稳定(时强时弱、易猝灭)。建议探针分装避光保存在-20°C冰箱中,避免反复冻融。

6.阳性对照荧光弱

阳性对照Rosup通常浓度为100 μM。通常刺激后30 min-4 h可以观察到显著的活性氧水平升高。对于不同的细胞,活性氧阳性对照的效果可能有较大的差别。如果在刺激后30 min内观察不到ROS的升高,可延长诱导时间或适当提高Rosup的浓度。


已发表文章(部分)

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