Measuring Mitochondrial Transmembrane Potential by TMRE Staining.
Measuring Mitochondrial Transmembrane Potential by TMRE Staining.
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DOI：
10.1101/pdb.prot087361
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发表时间：
2016-12-01
影响因子：
--
通讯作者：
Waterhouse, Nigel J
中科院分区：
文献类型：
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作者：
Crowley, Lisa C;Christensen, Melinda E;Waterhouse, Nigel J
三磷酸腺苷（ATP）是新陈代谢的主要能量来源。线粒体通过氧化磷酸化过程提供大部分 ATP。该过程涉及带正电的质子穿过线粒体内膜的主动转移，从而产生净内部负电荷，称为线粒体跨膜电位（DeltaPsim）。然后，质子梯度被 ATP 合酶利用，通过融合二磷酸腺苷和游离磷酸盐来产生 ATP。健康线粒体的净负电荷保持在大约 -180 mV，可以通过用带正电荷的染料（例如四甲基罗丹明乙酯 (TMRE)）对细胞染色来检测。 TMRE 发出红色荧光，可通过流式细胞术或荧光显微镜检测，染色细胞中 TMRE 荧光的水平可用于确定细胞中线粒体 DeltaPsim 的高低。细胞色素 c 对于产生 DeltaPsim 至关重要，因为当它沿着电子传输链将电子从复合物 III 穿梭到复合物 IV 时，它会促进将质子泵入线粒体膜间空间。细胞色素 c 在细胞凋亡过程中从线粒体膜间隙释放到细胞质中。这削弱了其在复合物 III 和复合物 IV 之间穿梭电子的能力，并导致 DeltaPsim 快速耗散。因此，DeltaPsim 的缺失与细胞凋亡过程中细胞色素 c 的释放密切相关，并且通常用作细胞中细胞色素 c 释放的替代标记。
Adenosine triphosphate (ATP) is the main source of energy for metabolism. Mitochondria provide the majority of this ATP by a process known as oxidative phosphorylation. This process involves active transfer of positively charged protons across the mitochondrial inner membrane resulting in a net internal negative charge, known as the mitochondrial transmembrane potential (DeltaPsim). The proton gradient is then used by ATP synthase to produce ATP by fusing adenosine diphosphate and free phosphate. The net negative charge across a healthy mitochondrion is maintained at approximately -180 mV, which can be detected by staining cells with positively charged dyes such as tetramethylrhodamine ethyl ester (TMRE). TMRE emits a red fluorescence that can be detected by flow cytometry or fluorescence microscopy and the level of TMRE fluorescence in stained cells can be used to determine whether mitochondria in a cell have high or low DeltaPsim. Cytochrome c is essential for producing DeltaPsim because it promotes the pumping the protons into the mitochondrial intermembrane space as it shuttles electrons from Complex III to Complex IV along the electron transport chain. Cytochrome c is released from the mitochondrial intermembrane space into the cytosol during apoptosis. This impairs its ability to shuttle electrons between Complex III and Complex IV and results in rapid dissipation of DeltaPsim. Loss of DeltaPsim is therefore closely associated with cytochrome c release during apoptosis and is often used as a surrogate marker for cytochrome c release in cells.