Arnoult Damien
INSERM U 542, Université Paris-Sud, Hopital Paul Brousse, Batiment Lavoisier, 14 avenue Paul Vaillant Couturier, 94807 Villejuif cedex, France.
Methods. 2008 Mar;44(3):229-34. doi: 10.1016/j.ymeth.2007.11.003.
Following most cell death signals, pro-apoptotic Bcl-2 members as Bax and Bak are activated and oligomerize into the mitochondria outer membrane, triggering its permeabilization and release into the cytosol of soluble apoptogenic factors such as cytochrome c involved in caspase activation. Thus, in many studies focused on apoptosis, cytochrome c release within cells is frequently examined to assess Bax/Bak activation and mitochondrial outer membrane permeabilization. In addition, cytochrome c release can also be investigated in vitro in functional mitochondria that have been isolated from cultured cells, offering a number of advantages. Here, protocols for measuring cytochrome c release from intact cells as well as from isolated mitochondria is detailed. Finally, assays to investigate Bax/Bak activation and olimerization are also presented.
在大多数细胞死亡信号作用下，促凋亡的Bcl-2家族成员如Bax和Bak被激活并在线粒体外膜寡聚化，引发线粒体外膜通透性改变，并将诸如参与半胱天冬酶激活的细胞色素c等可溶性促凋亡因子释放到细胞质中。因此，在许多聚焦于细胞凋亡的研究中，经常检测细胞内细胞色素c的释放，以评估Bax/Bak的激活及线粒体外膜通透性改变。此外，也可以在从培养细胞中分离出的功能性线粒体中体外研究细胞色素c的释放，这具有诸多优势。在此，详细介绍了测量完整细胞以及分离线粒体中细胞色素c释放的实验方案。最后，还介绍了用于研究Bax/Bak激活和寡聚化的检测方法。
