Abstract
Based on our hypothesis that myotubes exhibit a bistable response to insulin, here we present a protocol for finely measuring Akt phosphorylation in single myotubes under insulin stimulation. We describe steps to stably express a Förster resonance energy transfer (FRET)-based Akt biosensor in C2C12-derived myotubes and perform single-cell FRET imaging. This protocol highlights its potential for precision medicine in analyzing protein phosphorylation dynamics at the single-cell level. For complete details on the use and execution of this protocol, please refer to Akhtar et al.1.