In vivo and in vitro determination of cell death markers in neurons.
In vivo and in vitro determination of cell death markers in neurons.
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神经元细胞死亡标志物的体内和体外测定。
DOI：
10.1007/978-1-61779-328-8_2
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发表时间：
2011
期刊：
影响因子：
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通讯作者：
Pasinelli,Piera
中科院分区：
文献类型：
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作者：
Naniche,Nicole;Sau,Daniela;Pasinelli,Piera
线粒体是细胞死亡的关键调节因子。线粒体膜含有维持代谢稳态和满足细胞能量需求的必需酶复合物（Tait和Green， Nat Rev Mol cell Biol 11:21 - 632, 2010和Galluzzi等人，凋亡12:803-813,2007）。因此，线粒体外膜或内膜的任何扰动都可能导致关键离子和代谢蛋白（即ADP/ATP交换）的正常通量中断，最终导致细胞死亡。除了维持细胞活力外，线粒体在细胞程序性死亡的开始中起着关键作用。作为细胞死亡过程的启动者，关键的线粒体蛋白[细胞色素C （Cyt C）是其中研究最充分的一种]在细胞早期死亡事件中从膜间空间释放，最终导致半胱天冬酶激活。Cyt C的释放是细胞死亡的关键步骤（Tait and Green, Nat Rev Mol Cell Biol 11:621-632, 2010）。因此，测量Cyt C释放可以提供关于细胞死亡信号的重要信息。针对Cyt C的免疫标记也可以很容易地读出线粒体完整性，允许同时识别线粒体活力（和/或损伤）和细胞内死亡过程的启动。在本章中，我们使用Cyt C作为线粒体完整性和细胞死亡的双重标记，并回顾了几种测量Cyt C定位到完整线粒体及其释放到细胞质中的方法。目标是提供一系列的分析，结合起来，提供线粒体活力和细胞内细胞死亡过程激活之间关系的定性和定量分析。免疫荧光，免疫印迹，和ELISA测量的细胞C的详细讨论。
Mitochondria are key regulators of cellular death. The mitochondrial membranes contain essential enzyme complexes for maintaining metabolic homeostasis and meeting the energy requirements of the cell (Tait and Green, Nat Rev Mol Cell Biol 11:621–632, 2010 and Galluzzi et al., Apoptosis 12:803–813, 2007). Thus, any perturbation of outer or inner mitochondrial membranes can lead to disruptions in the normal fluxes of key ions and metabolic proteins (i.e., ADP/ATP exchange), leading to eventual cellular death. In addition to maintaining cellular viability, mitochondria play a critical role in the initiation of programmed cell death. As initiators of the cell death process, key mitochondrial proteins [Cytochrome C (Cyt C) one of the most well-studied among them] are released from the intermembrane space during early cell death events eventually leading to caspase activation. Release of Cyt C is a crucial step during cellular death (Tait and Green, Nat Rev Mol Cell Biol 11:621–632, 2010). Therefore, the measurement of Cyt C release can give vital information about cell death signaling. Immunolabeling against Cyt C can give an easy readout of mitochondrial integrity as well, allowing for simultaneous identification of mitochondrial viability (and/or damage) and initiation of intracellular death processes. In this chapter, we use Cyt C as a dual marker of mitochondrial integrity and cell death and review several protocols to measure Cyt C localization into intact mitochondria and its release into the cytosol. The goal is to offer an array of assays that, combined, provide both qualitative and quantitative analysis of the relationship between mitochondrial viability and activation of an intracellular cell death process. Immunofluorescence, Western blot, and ELISA measurements of Cyt C as are discussed in detail.