Protocol for multiplexed quantification of fluorescent biosensor FRET efficiency using barcoded cells

Genetically encoded fluorescent biosensors frequently use Förster resonance energy transfer (FRET) between donor and acceptor fluorescent proteins (FPs) as readouts for monitoring molecular activities in live cells. Here, we present a protocol for determining FRET efficiency through the simultaneous imaging of multiple FRET biosensors alongside donor and acceptor FPs using spectrally orthogonal fluorescent cell barcodes. We describe steps for culturing and transfecting cells, acquiring and analyzing images, and calculating FRET efficiency. This approach facilitates simultaneous analysis of multiple FRET biosensors. For complete details on the use and execution of this protocol, please refer to Wu et al.¹.