Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells
Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells
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DOI：
10.1016/s0167-4889(98)00088-3
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发表时间：
1998-09-16
影响因子：
5.1
通讯作者：
Wu, SL
中科院分区：
文献类型：
--
作者：
Rottenberg, H;Wu, SL
线粒体膜电位，原位，是线粒体功能和功能障碍的重要指标。由于最近对线粒体在信号传导、细胞损伤和细胞死亡中的作用的兴趣，需要一种方便、敏感和准确的方法来原位测量异质细胞群体中的线粒体膜电位Delta psi(m)。我们采用了一种流式细胞术方法来定量测量Delta Psi(m)，该方法利用亲脂、阳离子、荧光探针3,3'-二己基氯碳氰酸碘(DiOC（6)(3)）。我们开发了一种新的方案，其中细胞与非常低的染料浓度（< 1 nM）平衡。只有在这些条件下，细胞荧光似乎与δ Psi(m)的大小相关，这一点从荧光对低浓度解偶联剂、离子载体和线粒体质子泵抑制剂的敏感性可见一斑。质膜电位δ Psi(p)的大小也影响细胞荧光，并概述了纠正这种影响的程序。这种方法提供了一个明显的优势比现有的方法估计δ psi(m)流式细胞术。0167-4889/98/$ -见封面(C) 1998 Elsevier Science B.V.版权所有
Mitochondrial membrane potential, in situ, is an important indicator of mitochondrial function and dysfunction. Because of recent interest in the role of mitochondria in signaling, cell injury and cell death, there is a need for a convenient, sensitive and accurate method for the measurement of the mitochondrial membrane potential, Delta psi(m), in situ, in a heterogeneous cell population. We have adapted a flow cytometry method for the quantitative measurement of Delta Psi(m) which utilizes the lipophilic, cationic, fluorescent probe 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)(3)). We developed a new protocol in which cells are equilibrated with very low dye concentrations (< 1 nM). Only under these condition, the cell fluorescence appears to be correlated with the magnitude of Delta Psi(m), as evident from the sensitivity of the fluorescence to low concentrations of uncouplers, ionophores and inhibitors of the mitochondrial proton pumps. The magnitude of the plasma membrane potential, Delta Psi(p), also affects cell fluorescence, and a procedure that corrects for this effect is outlined. This method offers a distinct advantage over existing methods for estimation of Delta psi(m) by flow cytometry. 0167-4889/98/$ - see front matter (C) 1998 Elsevier Science B.V. All rights reserved.