Characterizing the Electron Transport Chain: Functional Approach Using Extracellular Flux Analyzer on Mouse Tissue Samples.

The Seahorse Extracellular Flux Analyzer enables the high-throughput characterization of oxidative phosphorylation capacity based on the electron transport chain organization and regulation with relatively small amount of material. This development over the traditional polarographic Clark-type electrode approaches make it possible to analyze the respiratory features of mitochondria isolated from tissue samples of particular animal models. Here we provide a description of an optimized approach to carry out multi-well measurement of O2 consumption, with the Agilent Seahorse XFe96 analyzer on mouse brain and muscles to determine the tissue-specific oxidative phosphorylation properties. Protocols include the preparation of the tissue samples, isolation of mitochondria, and analysis of their function; in particular, the preparation and optimization of the reagents and samples.