A method for analyzing protein-protein interactions in the plasma membrane of live B cells by fluorescence resonance energy transfer imaging as acquired by total internal reflection fluorescence microscopy.

For more than a decade, fluorescence resonance energy transfer (FRET) imaging methods have been developed to study dynamic interactions between molecules at the nanometer scale in live cells. Here, we describe a protocol to measure FRET by the acceptor-sensitized emission method as detected by total internal reflection fluorescence (TIRF) imaging to study the interaction of appropriately labeled plasma membrane-associated molecules that regulate the earliest stages of antigen-mediated signaling in live B lymphocytes. This protocol can be adapted and applied to many cell types where there is an interest in understanding signal transduction mechanisms in live cells.