Assessing Mitochondrial Bioenergetics in Isolated Mitochondria from Various Mouse Tissues Using Seahorse XF96 Analyzer.
Working with isolated mitochondria is the gold standard approach to investigate the function of the electron transport chain in tissues, free from the influence of other cellular factors. In this chapter, we outline a detailed protocol to measure the rate of oxygen consumption (OCR) with the high-throughput analyzer Seahorse XF96. More importantly, this protocol wants to provide practical tips for handling many different samples at once, and take a real advantage of using a high-throughput system. As a proof of concept, we have isolated mitochondria from brain, heart, liver, muscle, kidney, and lung of a wild-type mouse, and measured basal respiration (State II), ADP-stimulated respiration (State III), non-ADP-stimulated respiration (State IVo), and FCCP-stimulated respiration (State IIIu) using respiratory substrates specific to the respiratory chain complex I (RCCI) and complex II (RCCII). Mitochondrial purification and Seahorse runs were performed in less than eight working hours.
['Iuso|Arcangela|A|', 'Repp|Birgit|B|', 'Biagosch|Caroline|C|', 'Terrile|Caterina|C|', 'Prokisch|Holger|H|']
D000818:Animals D002458:Cell Fractionation D004734:Energy Metabolism D057166:High-Throughput Screening Assays D051379:Mice D008928:Mitochondria D009928:Organ Specificity D010101:Oxygen Consumption D013223:Statistics as Topic
Mouse tissues Seahorse XF96 High-throughput Mitochondrial coupling assay Respiratory control ratio (RCR)