Nonnenmacher Yannic, Palorini Roberta, Hiller Karsten
Department of Bioinformatics and Biochemistry, Braunschweig Integrated Center of Systems Biology (BRICS), Technische Universität Braunschweig, Braunschweig, Germany.
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy.
Methods Mol Biol. 2019;1862:137-149. doi: 10.1007/978-1-4939-8769-6_10.
In this chapter, we present an experimental protocol for the targeted metabolic profiling of full cells and mitochondria in selectively permeabilized cells. Mitochondria of adherent cell cultures are made accessible by the addition of digitonin-a compound that selectively permeabilizes the cytosolic membrane without affecting mitochondrial integrity. The generated in situ mitochondria are subsequently used in a stable isotope labeling assay in which their metabolic fluxes can be analyzed without any interfering influence originating from cytosolic components. The protocol is complemented by oxygen consumption measurements of permeabilized cells on a Seahorse XF instrument. The additional data on mitochondrial respiration can be used to validate the functionality of mitochondria in the applied setup but are also a valuable add-on to the stable isotope labeling data.
在本章中，我们介绍了一种用于选择性通透细胞中全细胞和线粒体靶向代谢谱分析的实验方案。通过添加洋地黄皂苷，使贴壁细胞培养物中的线粒体可被检测到，洋地黄皂苷是一种能选择性通透细胞质膜而不影响线粒体完整性的化合物。随后，将原位生成的线粒体用于稳定同位素标记分析，在该分析中，无需任何来自细胞质成分的干扰影响，即可分析其代谢通量。该方案通过在Seahorse XF仪器上对通透细胞进行耗氧量测量得以补充。关于线粒体呼吸的额外数据可用于验证所应用设置中线粒体的功能，但也是对稳定同位素标记数据的宝贵补充。
