Almazov National Medical Research Centre, Saint Petersburg, Russia.
Istituto Auxologico Italiano IRCCS, Center for Cardiac Arrhythmias of Genetic Origin and Laboratory of Cardiovascular Genetics, Milan, Italy.
Methods Mol Biol. 2023;2644:3-14. doi: 10.1007/978-1-0716-3052-5_1.
Mitochondrial respiration is an essential component of cellular metabolism. It is a process of energy conversion through enzymatically mediated reactions, the energy of taken-up substrates transformed to the ATP production. Seahorse equipment allows to measure oxygen consumption in living cells and estimate key parameters of mitochondrial respiration in real-time mode. Four key mitochondrial respiration parameters could be measured: basal respiration, ATP-production coupled respiration, maximal respiration, and proton leak. This approach demands the application of mitochondrial inhibitors-oligomycin to inhibit ATP synthase, FCCP-to uncouple the inner mitochondrial membrane and allow maximum electron flux through the electron transport chain, rotenone, and antimycin A to inhibit complexes I and III, respectively. This chapter describes two protocols of seahorse measurements performed on iPSC-derived cardiomyocytes and TAZ knock-out C2C12 cell line.
线粒体呼吸是细胞代谢的一个重要组成部分。它是一个通过酶介导的反应将底物的能量转化为 ATP 生成的能量转换过程。 Seahorse 仪器可以测量活细胞中的耗氧量,并实时估计线粒体呼吸的关键参数。可以测量四个关键的线粒体呼吸参数:基础呼吸、与 ATP 产生偶联的呼吸、最大呼吸和质子漏。这种方法需要应用线粒体抑制剂寡霉素来抑制 ATP 合酶,应用 FCCP 来解偶联线粒体内膜,使电子通过电子传递链的最大流速,应用鱼藤酮和安密妥 A 分别抑制复合物 I 和 III。本章描述了两种在 iPSC 衍生的心肌细胞和 TAZ 敲除 C2C12 细胞系上进行 Seahorse 测量的方案。