Dave Zankruti, Byfield Maya, Bossy-Wetzel Ella
Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, 4000 Central Florida Blvd., Orlando, FL 32816, USA.
Methods. 2008 Dec;46(4):319-23. doi: 10.1016/j.ymeth.2008.10.019. Epub 2008 Oct 26.
Mitochondria play a pivotal role in the regulation of apoptosis. An imbalance in apoptosis can lead to disease. Unscheduled apoptosis has been linked to neurodegeneration while inhibition of apoptosis can cause cancer. An early and key event during apoptosis is the release of factors from mitochondria. In apoptosis the mitochondrial outer membrane becomes permeable, leading to release of apoptogenic factors into the cytosol. One such factor, cytochrome c, is an electron carrier of the respiratory chain normally trapped within the mitochondrial intermembrane space. Many apoptotic studies investigate mitochondrial outer membrane permeabilization (MOMP) by monitoring the release of cytochrome c. Here, we describe three reliable techniques that detect cytochrome c release from mitochondria, through subcellular fractionation or immunocytochemistry and fluorescence microscopy, or isolated mitochondria and recombinant Bax and t-Bid proteins in vitro. These techniques will help to identify mechanisms and characterize factors regulating MOMP.
线粒体在细胞凋亡的调控中起着关键作用。细胞凋亡失衡会导致疾病。非程序性细胞凋亡与神经退行性变有关，而细胞凋亡的抑制则会引发癌症。细胞凋亡过程中的一个早期关键事件是线粒体释放因子。在细胞凋亡过程中，线粒体外膜变得通透，导致凋亡因子释放到细胞质中。细胞色素c就是这样一种因子，它是呼吸链的电子载体，通常被困在线粒体内膜间隙中。许多细胞凋亡研究通过监测细胞色素c的释放来研究线粒体外膜通透性改变（MOMP）。在此，我们描述了三种可靠的技术，通过亚细胞分级分离、免疫细胞化学和荧光显微镜，或体外分离的线粒体以及重组Bax和t-Bid蛋白，来检测细胞色素c从线粒体的释放。这些技术将有助于确定调控MOMP的机制并对相关因子进行特性描述。
