Fluorescence-Based Microplate Assays for In Vitro Assessment of Mitochondrial Toxicity, Metabolic Perturbation, and Cellular Oxygenation

James Hynes, Conn Carey, Yvonne Will
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引用次数: 8

Abstract

High-throughput in vitro cell metabolism assays are of particular use for identification and delineation of mitochondrial toxicity and related metabolic perturbation. Here, a panel of fluorescence-based metabolism assays are described for measuring oxygen consumption, glycolytic flux, and cellular oxygenation. They can be applied to analysis of both isolated mitochondria and cell models. Sample data are presented illustrating how these protocols can be used to examine drug treatment, the interplay between oxidative and glycolytic ATP generation, and the impact of cell oxygenation on this balance. Descriptions are provided on how these measurements can be applied to 3D systems and how they can be multiplexed with other relevant metabolic readouts. Mitochondrial isolation and cell permeabilization protocols are also provided. © 2016 by John Wiley & Sons, Inc.

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荧光微孔板法测定线粒体毒性,代谢扰动和细胞氧化的体外评估
高通量体外细胞代谢测定特别用于鉴定和描述线粒体毒性和相关代谢扰动。在这里,一组基于荧光的代谢测定被描述为测量氧气消耗,糖酵解通量和细胞氧合。它们可以应用于分离线粒体和细胞模型的分析。样本数据展示了这些方案如何用于检查药物治疗,氧化和糖酵解ATP生成之间的相互作用,以及细胞氧合对这种平衡的影响。描述了如何将这些测量应用于3D系统,以及如何将它们与其他相关的代谢读数进行多路复用。还提供了线粒体分离和细胞渗透方案。©2016 by John Wiley &儿子,Inc。
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