Ferlini Cristiano, Scambia Giovanni
Laboratory of Antineoplastic Pharmacology, Department of Obstetrics and Gynaecology, Catholic University of the Sacred Heart, Rome, Italy.
Nat Protoc. 2007;2(12):3111-4. doi: 10.1038/nprot.2007.397.
Apoptosis plays a pivotal role in the regulation of cell turnover, and a defect or an excess of apoptosis has been implicated in several human diseases. Apoptosis is activated from an extracellular death signal, or from an internal pathway starting from the endoplasmatic reticulum or the mitochondria. To investigate the mitochondrial compartment during apoptosis, we have established a protocol using fluorochromes and flow cytometry to probe the structure and function of mitochondria kinetically. The protocol could be applied to whole cells or to isolated mitochondria. In the first case, cells are counterstained with ethidium bromide (EB) to evaluate plasma membrane function. The presence of the electrochemical gradient in the mitochondria is probed with Rhodamine123 (Rh123), whereas the structure and the integrity of mitochondria are assessed using 10-N-nonyl-acridine orange (NAO). Not considering the time requested for cell/mitochondria preparation and the activation of apoptosis, the protocol lasts <1 h.
细胞凋亡在细胞更新的调节中起关键作用，细胞凋亡的缺陷或过度与多种人类疾病有关。细胞凋亡可由细胞外死亡信号激活，也可由始于内质网或线粒体的内部途径激活。为了在细胞凋亡过程中研究线粒体区室，我们建立了一种使用荧光染料和流式细胞术动态探测线粒体结构和功能的方法。该方法可应用于完整细胞或分离的线粒体。在第一种情况下，用溴化乙锭（EB）对细胞进行复染以评估质膜功能。用罗丹明123（Rh123）探测线粒体中电化学梯度的存在，而使用10-N-壬基吖啶橙（NAO）评估线粒体的结构和完整性。不考虑细胞/线粒体制备和细胞凋亡激活所需的时间，该方法耗时不到1小时。
