Flow cytometric FRET analysis of protein interaction.

Investigation of protein-protein interactions in situ in living or intact cells gains expanding importance as structure/function relationships proposed from bulk biochemistry and molecular modeling experiments require demonstration at the cellular level. Fluorescence resonance energy transfer (FRET)-based methods are excellent tools for determining proximity and supramolecular organization of biomolecules at the cell surface or inside the cell. This could well be the basis for the increasing popularity of FRET; in fact, the number of publications exploiting FRET has doubled in the past 5 years. In this chapter, we intend to provide a generally useable protocol for measuring FRET in flow cytometry. After a concise theoretical introduction, recipes are provided for successful labeling techniques and measurement approaches. The simple, quenching-based population-level measurement; the classic ratiometric, intensity-based technique providing cell-by-cell actual FRET efficiencies, and a more advanced version of the latter, allowing for cell-by-cell autofluorescence correction, are described. Finally, points of caution are given to help design proper experiments and critically interpret the results.