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Journal Article

Measuring Bioenergetics in T Cells Using a Seahorse Extracellular Flux Analyzer


Pearce,  Erika L.
Department Immunometabolism, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

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van der Windt, G. J., Chang, C., & Pearce, E. L. (2016). Measuring Bioenergetics in T Cells Using a Seahorse Extracellular Flux Analyzer. Current Protocols in Immunology, 113, 16B1.3-16B.14. doi:10.1002/0471142735.im0316bs113.

Cite as: https://hdl.handle.net/21.11116/0000-0006-4C15-E
This unit contains several protocols to determine the energy utilization of T cells in real‐time using a Seahorse Extracellular Flux Analyzer (http://www.seahorsebio.com). The advantages to using this machine over traditional metabolic assays include the simultaneous measurement of glycolysis and mitochondrial respiration, in real‐time, on relatively small numbers of cells, without any radioactivity. The Basic Protocol describes a standard mitochondrial stress test on the XFe96, which yields information about oxidative phosphorylation and glycolysis, two energy‐generating pathways. The alternate protocols provide examples of adaptations to the Basic Protocol, including adjustments for the use of the XFe24. A protocol for real‐time bioenergetic responses to T cell activation allows for the analysis of immediate metabolic changes after T cell receptor stimulation. Specific substrate utilization can be determined by the use of differential assay media, or the injection of drugs that specifically affect certain metabolic processes. Accurate cell numbers, purity, and viability are critical to obtain reliable results.