Protocol for pH correction of FRET-based fluorescent biosensor imaging in dissociated Drosophila neurons

Fluorescent biosensors are sensitive to intracellular pH fluctuations, which can shift fluorophore properties and compromise accurate metabolite quantification. We present a protocol for pH correction in FRET-based metabolic biosensor imaging using dissociated Drosophila larval neurons. We describe steps for integrating cytosolic pH measurements with R-based post hoc correction of FRET ratios. We also detail procedures for brain dissection, tissue dissociation, primary neuron culture, and live-cell imaging. This protocol enables accurate metabolic biosensor readouts at single-cell resolution. For complete details on the use and execution of this protocol, please refer to Price et al. 1