Flow Cytometric FRET Analysis of Protein Interactions.

In the past decades, investigation of protein-protein interactions in situ in living or intact cells has gained expanding importance as structure/function relationships proposed from bulk biochemistry and molecular modeling experiments required confirmation at the cellular level. Förster (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 exploded since the turn of the millennium. Interestingly, most applications are microscope-based, and only a fraction employs flow cytometry, even though the latter offers great statistical power owed to the potentially huge number of individually measured cells. However, with the increased availability of multi-laser flow cytometers, strategies to obtain absolute FRET efficiencies can now be relatively facilely implemented. In this chapter, we intend to provide generally useable protocols 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. An Excel macro pre-loaded with spectral data of the most commonly used fluorophores is also provided for easy calculation of average FRET efficiencies. Finally, points of caution are given to help design proper experiments and critically interpret the results.