Protocol for characterizing cell motility and extracellular signal-regulated kinase dynamics in epithelial monolayers using FRET imaging data

Extracellular signal-regulated kinase (ERK) activity waves regulate critical processes like wound healing and bacterial pathogen dissemination by altering host cell motility and biomechanics. Here, we present a protocol for fluorescence resonance energy transfer (FRET) imaging of ERK activity in the nuclei of epithelial cells in a monolayer using an ERK biosensor. Moreover, we outline all image processing steps for the spatiotemporal quantification of single-cell ERK oscillations and wave propagation. Modifications, especially with respect to FRET imaging, may be necessary if different ERK biosensors are used. For complete details on the use and execution of this protocol, please refer to Hundsdorfer et al.¹.