1、 活性氧检测试剂盒活性氧检测试剂盒 产品编号 产品名称 包装 S0033 活性氧检测试剂盒 100次 产品简介：产品简介：活性氧检测试剂盒(Reactive Oxygen Species Assay Kit)是一种利用荧光探针DCFH-DA进行活性氧检测的试剂盒。DCFH-DA本身没有荧光，可以自由穿过细胞膜，进入细胞内后，可以被细胞内的酯酶水解生成DCFH。而DCFH不能通透细胞膜，从而使探针很容易被装载到细胞内。细胞内的活性氧可以氧化无荧光的DCFH生成有荧光的DCF。检测DCF的荧光就可以知道细胞内活性氧的水平。本试剂盒提供了活性氧阳性对照试剂Rosup，以便于活性氧的检测。Rosup是
2、一种混合物(compound mixture),浓度为50mg/ml。本试剂盒本底低，灵敏度高，线性范围宽，使用方便。本试剂盒可以测定100-500个样品。包装清单：包装清单：产品编号 产品名称 包装 S0033-1 DCFH-DA(10mM)0.1ml S0033-2 活性氧阳性对照(Rosup,50mg/ml)1ml 说明书 1份 保存条件：保存条件：-20保存，一年有效。注意事项：注意事项：探针装载后，一定要洗净残余的未进入细胞内的探针，否则会导致背景较高。探针装载完毕并洗净残余探针后，可以进行激发波长的扫描和发射波长的扫描，以确认探针的装载情况是否良好。DCF的激发光谱和发射光谱请参考
3、下页图谱。尽量缩短探针装载后到测定所用的时间(刺激时间除外)，以减少各种可能的误差。为了您的安全和健康，请穿实验服并戴一次性手套操作。使用说明：使用说明：1.装载探针装载探针 对于刺激时间较短(通常为2小时以内)的细胞，先装载探针，后用活性氧阳性对照或自己感兴趣的药物刺激细胞。对于细胞刺激时间较长(通常为6小时以上)的细胞，先用活性氧阳性对照或自己感兴趣的药物刺激细胞，后装载探针。原位装载探针：原位装载探针：本方法仅适用于贴壁培养细胞。按照1:1000用无血清培养液稀释DCFH-DA，使终浓度为10微摩尔/升。去除细胞培养液，加入适当体积稀释好的DCFH-DA。加入的体积以能充分盖住细胞为宜，
4、通常对于六孔板的一个孔加入稀释好的DCFH-DA不少于1毫升。37细胞培养箱内孵育20分钟。用无血清细胞培养液洗涤细胞三次，以充分去除未进入细胞内的DCFH-DA。通常活性氧阳性对照在刺激细胞20-30分钟后可以显著提高活性氧水平。收集细胞后收集细胞后装载探针：装载探针：按照1:1000用无血清培养液稀释DCFH-DA，使终浓度为10微摩尔/升。细胞收集后悬浮于稀释好的DCFH-DA中，细胞浓度为一百万至二千万/毫升，37细胞培养箱内孵育20分钟。每隔3-5分钟颠倒混匀一下，使探针和细胞充分接触。用无血清细胞培养液洗涤细胞三次，以充分去除未进入细胞内的DCFH-DA。直接用活性氧阳性对照或自己
5、感兴趣的药物刺激细胞，或把细胞等分成若干份后刺激细胞。通常活性氧阳性对照在刺激细胞20-30分钟后可以显著提高活性氧水平。说明说明：仅在阳性对照孔中加入Rosup作为阳性对照，其余孔不必加入Rosup。2.检测检测 对于原位装载探针的样品可以用激光共聚焦显微镜直接观察，或收集细胞后用荧光分光光度计、荧光酶标仪或流式细胞仪检测。对于收集细胞后装载探针的样品可以用荧光分光光度计、荧光酶标仪或流式细胞仪检测，用激光共聚焦显微镜直接观察也可以。3.参数设置参数设置 使用488nm激发波长，525nm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
6、设置检测DCF。DCF的激发光谱和发射光谱参考下图。4.其它说明其它说明 阳性对照可以按照1:1000的比例使用。例如装载好探针的细胞共1毫升，可以加入1微升的阳性对照刺激。通常刺激后20-30分钟内可以观察到非常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分钟内观察不到活性氧的升高，可以适当提高活性氧阳性对照的浓度。如果活性氧升高得过快，可以适当降低活性氧阳性对照的浓度。另外，对于某些细胞，如果发现没有刺激的阴性对照细胞荧光也比较强，可以按照1:2000-1:5000稀释DCFH-DA，使装载探针时DCFH-DA的浓度为2-5微摩尔/升。探针装
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