Detecting Förster resonance energy transfer in living cells by conventional and spectral flow cytometry

Assays based on Förster resonance energy transfer (FRET) can be used to study many processes in cell biology. Although this is most often done with microscopy for fluorescence detection, we report two ways to measure FRET in living cells by flow cytometry. Using a conventional flow cytometer and the "3-cube method" for intensity-based calculation of FRET efficiency, we measured the enzymatic activity of specific kinases in cells expressing a genetically-encoded reporter. For both AKT and protein kinase A, the method measured kinase activity in time-course, dose-response, and kinetic assays. Using the Cytek Aurora spectral flow cytometer, which applies linear unmixing to emission measured in multiple wavelength ranges, FRET from the same reporters was measured with greater single-cell precision, in real time and in the presence of other fluorophores. Results from gene-knockout studies suggested that spectral flow cytometry might enable the sorting of cells on the basis of FRET. The methods we present provide convenient and flexible options for using FRET with flow cytometry in studies of cell biology.