Protocol to assess bioenergetics and mitochondrial fuel usage in murine autoreactive immunocytes using the Seahorse Extracellular Flux Analyzer

Efficient metabolism, or the means by which cells produce energy resources, is critical for proper effector function. Here, we present a protocol for examining the bioenergetics and mitochondrial fuel utilization of primary murine autoreactive immunocytes using cellular metabolism-modulating drugs. We describe steps for plate calibration, isolation of primary immunocytes, and Seahorse assay plate preparation. We then detail procedures for performing the XF Cell Mito Stress Test followed by bioenergetics calculations and statistics. For complete details on the use and execution of this protocol, please refer to Wilson et al.¹.