Protocol for pH correction of FRET-based fluorescent biosensor imaging in dissociated Drosophila neurons
Matthew S Price1, Elham Rastegari2, Kartik Venkatachalam3
1Department of Integrative Biology and Pharmacology, McGovern Medical School at the University of Texas Health Sciences Center (UTHealth), Houston, TX, USA; Neuroscience Graduate Program, The University of Texas MD Anderson Cancer Center UTHealth Houston Graduate School of Biomedical Sciences, Houston, TX, USA.
Abstract:
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.
