Protocol to quantify desmoglein 3 tension at cell-cell adhesions using FRET-based tension sensors

Desmoglein 3 (Dsg3) is a crucial component of cell-cell junctions, contributing to mechanotransduction and maintaining tissue integrity. Here, we present a protocol to quantify tension at keratinocyte cell-cell adhesion sites using a Dsg3 fluorescence resonance energy transfer (FRET)-based tension sensor. We describe steps for increasing viral titer by amplifying adenovirus in HEK293A cells and optimizing infection conditions to ensure uniform expression of FRET signals. We detail procedures for live-cell imaging of keratinocytes and image analysis to quantify the FRET ratio. For complete details on the use and execution of this protocol, please refer to Jin et al.¹.