Flow cytometric analysis of CFP–YFP FRET as a marker for in vivo protein–protein interaction

The technique of observing fluorescence resonance energy transfer (FRET) between a cyan fluorescent protein (CFP) fusion protein and a yellow fluorescent protein (YFP) fusion protein has been used in numerous studies as a reliable indicator of protein–protein interaction. Moreover, because CFP and YFP fusions generally retain activity and maintain their normal subcellular localizations, the detection of CFP–YFP FRET in live cells typically reflects the steady-state association of functional proteins in their native states. Although CFP–YFP FRET can also be monitored by fluorimetry and fluorescence microscopy, the ability of flow cytometry to rapidly acquire multiparameter fluorescence data on a large number of individual cells provides several important advantages. The analysis of CFP–YFP FRET on a cell-by-cell basis allows for a sensitive and highly rigorous assessment of protein interaction, and the large number of cells that can be examined by flow cytometry provides for a high degree of statistical confidence. In addition, simple, yet stringent, gating-based analyses of FRET can be performed on a flow cytometer simultaneously with data collection, allowing FRET-based cell sorting that can be used in high-throughput screens to identify interacting proteins. As a brief review of flow cytometric CFP–YFP FRET analysis, this article outlines a typical instrument configuration, describes the required controls, explains how gating is performed, and discusses the basic physical principles behind FRET as they relate to the successful design and interpretation of protein interaction experiments.