Protocol for quantifying LC3B FRET biosensor activity in living cells using a broad-to-sensitive data analysis pipeline

Here, we present a protocol to comprehensively quantify autophagy initiation using the readout of the microtubule associated protein 1 light chain 3 beta (LC3B) Förster's resonance energy transfer (FRET) biosensor. We describe steps for cell seeding, transfection, FRET/FLIM (fluorescence lifetime imaging microscopy) imaging, and image analysis. This protocol can be useful in any physiology- or disease-related paradigm where the LC3B biosensor can be expressed to determine whether autophagy has been initiated or is stalled. The analysis pipeline presented here can be applied to any other genetically encoded FRET sensor imaged using FRET/FLIM. For complete details on the use and execution of this protocol, please refer to Gökerküçük et al.¹.