Protocol to monitor live-cell, real-time, mitochondrial respiration in mouse muscle cells using the Resipher platform.

Mitochondrial function is typically assessed by measuring oxygen consumption at a given time point. However, this approach cannot monitor respiratory changes that occur over time. Here, we present a protocol to measure mitochondrial respiration in freshly isolated muscle stem cells, primary skeletal muscle, and immortalized C2C12 myoblasts in real time using the Resipher platform. We describe steps for preparing and plating cells, performing media changes, setting up the software and device, and analyzing data. This method can be adapted to other cell types. For complete details on the use and execution of this protocol, please refer to Triolo et al.¹.