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检测线粒体呼吸作为细胞代谢和功能的指标。

Assaying Mitochondrial Respiration as an Indicator of Cellular Metabolism and Fitness.

机构信息

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.

出版信息

Methods Mol Biol. 2023;2644:3-14. doi: 10.1007/978-1-0716-3052-5_1.

Abstract

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.

摘要

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。

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