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.
Keywords: 3D culture; cellular oxygenation; glycolysis; mitochondria; oxygen consumption; toxicity.
Copyright © 2016 John Wiley & Sons, Inc.