Crowley Lisa C, Christensen Melinda E, Waterhouse Nigel J
Apoptosis and Cytotoxicity Laboratory, Mater Research, Translational Research Institute, Woolloongabba, Brisbane, Queensland 4102, Australia.
Flow Cytometry and Imaging, QIMR Berghofer Medical Research Institute, Herston, Brisbane, Queensland 4006, Australia.
Cold Spring Harb Protoc. 2016 Dec 1;2016(12):2016/12/pdb.prot087361. doi: 10.1101/pdb.prot087361.
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 (ΔΨm). 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 ΔΨm. Cytochrome c is essential for producing ΔΨm 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 ΔΨm. Loss of ΔΨm is therefore closely associated with cytochrome c release during apoptosis and is often used as a surrogate marker for cytochrome c release in cells.
三磷酸腺苷（ATP）是新陈代谢的主要能量来源。线粒体通过一种称为氧化磷酸化的过程提供大部分的ATP。这个过程涉及带正电荷的质子在线粒体内膜上的主动转运，导致净内部负电荷，即线粒体跨膜电位（ΔΨm）。然后质子梯度被ATP合酶利用，通过将二磷酸腺苷和游离磷酸融合来产生ATP。健康线粒体上的净负电荷维持在大约 -180 mV，可以通过用带正电荷的染料如四甲基罗丹明乙酯（TMRE）对细胞进行染色来检测。TMRE发出红色荧光，可以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的线粒体具有高或低的ΔΨm。细胞色素c对于产生ΔΨm至关重要，因为当它沿着电子传递链将电子从复合物III穿梭到复合物IV时，它促进质子泵入线粒体膜间隙。细胞色素c在细胞凋亡期间从线粒体膜间隙释放到细胞质中。这损害了它在复合物III和复合物IV之间穿梭电子的能力，并导致ΔΨm迅速消散。因此，ΔΨm的丧失与细胞凋亡期间细胞色素c的释放密切相关，并且经常被用作细胞中细胞色素c释放的替代标志物。
