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.

BIOCEV 1st Faculty of Medicine Charles University Vestec Czech Republic

Department of Hematology Charles University and General University Hospital Prague Czech Republic

Department of Leukemia The University of Texas MD Anderson Cancer Center Houston Texas USA

Department of Lymphoma and Myeloma The University of Texas MD Anderson Cancer Center Houston Texas USA

Department of Translational Molecular Pathology The University of Texas MD Anderson Cancer Center Houston Texas USA

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Vertical targeting of the PI3K/AKT pathway at multiple points is synergistic and effective for non-Hodgkin lymphoma

Impact of PIK3CA gain and PTEN loss on mantle cell lymphoma biology and sensitivity to targeted therapies