JC-1, but not DiOC(6)(3) or rhodamine 123, is a reliable fluorescent probe to assess Delta Psi changes in intact cells: Implications for studies on mitochondrial functionality during apoptosis
JC-1, but not DiOC(6)(3) or rhodamine 123, is a reliable fluorescent probe to assess Delta Psi changes in intact cells: Implications for studies on mitochondrial functionality during apoptosis
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DOI：
10.1016/s0014-5793(97)00669-8
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发表时间：
1997-07-07
期刊：
影响因子：
3.5
通讯作者：
Cossarizza, A
中科院分区：
文献类型：
--
作者：
Salvioli, S;Ardizzoni, A;Cossarizza, A
在 U937 人类细胞系中研究了用于线粒体膜电位 (Delta Psi) 细胞荧光分析的三种荧光探针的灵敏度和特异性。首先，研究了质膜在影响探针与线粒体结合中的作用，用高剂量的细胞外 KCI 使质膜去极化对 JC-1、DiOC(6)(3) 和罗丹明 123 (R123) 的负载没有立即影响，然而，在 KCI 存在下培养几个小时后，仅在细胞中观察到显着变化 其次，对能够破坏 Delta Psi 的试剂的影响进行了比较研究，而在细胞培养物中添加 FCCP 会导致 JC-1 和 DiOC(6)(3) 的荧光发射发生一致变化，但 R123 则不然，只有用 JC-1 染色的细胞才会对缬氨霉素产生反应。总的来说，我们的数据表明，JC-1 是使用流式细胞术分析 Delta Psi 变化的可靠探针，而其他探针则由于对质膜电位变化的高敏感性而表现出较低的灵敏度 (R123) 或非相干行为 [DiOC(6)(3)]。这些数据对那些使用对 Delta Psi 敏感性较低的荧光探针假设 Delta Psi 下降即使不是细胞凋亡的主要原因之一也是早期事件之一的研究提出了一些质疑。 (C) 1997 年欧洲生化学会联合会。
The sensitivity and specificity of three fluorescent probes used for cytofluorimetric analysis of mitochondrial membrane potential (Delta Psi) were studied in the U937 human cell line. First, the role of plasmamembrane in influencing the binding of the probes to mitochondria has been investigated, The depolarization of plasmamembrane with high doses of extracellular KCI had no immediate effects on the loading of JC-1, DiOC(6)(3) and rhodamine 123 (R123), However, after a few hours of culture in the presence of KCI, significant changes were observed only in cells stained with DiOC(6)(3), Second, a comparative study was performed concerning the effects of agents capable of collapsing Delta Psi, While adding FCCP to cell cultures resulted in consistent changes in the fluorescence emission of both JC-1 and DiOC(6)(3) - but not of R123 - only cells stained with JC-1 responded to valinomycin. On the whole, our data indicate that JC-1 is a reliable probe for analyzing Delta Psi changes with flow cytometry, while the others show a lower sensitivity (R123), or a non-coherent behaviour, due to a high sensitivity to changes in plasmamembrane potential [DiOC(6)(3)]. These data cast some doubts on those studies that, using fluorescent probes that have a low sensitivity to Delta Psi hypothesized that the fall in Delta Psi is one of the early events, if not one of the main causes, of apoptosis. (C) 1997 Federation of European Biochemical Societies.