如何使用JC-1检测线粒体膜电位

如何使用JC-1检测线粒体膜电位

博凌科为生物科技-为你线粒体膜电位的降低是凋亡的一个早期事件,5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide (JC-1)可用于流式细胞术检测线粒体膜电位的变化.正常细胞的线粒体膜电位电压较高,可形成JC-1的聚集体,发出红色荧光,而线粒体膜电位降低的细胞,则会形成JC-1单体,发出绿色荧光.材料：细胞悬液 完全细胞培养基 去极化药物：如0.1mM valinomycin或0.25mM carbonyl cyanide m-(trifluoromethoxy)phenylhydrazone (FCCP) in DMSO 1mg/ml JC-1储存液（溶于DMSO）,分装成小包装,在－20℃可保存1年.PBS 具有488nm激发光源的流式细胞仪,配备525－590nm带宽的滤镜.JC-1染色：收集2105的细胞,用预温到37℃的新鲜完全培养基调整至1ml.制备阳性对照（以去极化药物处理过的细胞）.标本和阳性对照的染色步骤相同.融化1管JC-1储存液,每1ml细胞悬液中通过边振荡边加的方式加入2.5L（终浓度：2.5ug/ml）.振荡细胞悬液直至标本形成均一的红紫色.JC-1在水相容易形成聚集体,所以需要边振荡边加.未用完的JC-1不要再保存至－20℃.37℃避光保存10分钟.加入2ml PBS至细胞悬液中,500g室温离心5分钟,去上清.以0.3ml PBS重悬细胞.上机取样,注意获取细胞的速度不要250至300细胞/秒.Fig1 HL-60 cells,(A) control and (B) depolarized (treated with 100 nM valinomycin),stained with 2.5 g/ml JC-1.Note the shift to the bottom and to the right by cells with depolarized mitochondria.Fig2 After incubation with 2.5 g/ml JC-1 at 37C,peripheral blood mononulear cells usually show a single population of monocytes,while here lymphocytes give rise to two separate peaks.This could indicate the presence of a functional heterogeneity within lymphocytes (A).Such a phenomenon,however,is not always present.Treating cells with valinomycin results in a loss of orange signal either from lymphocytes or from monocytes (B).