Detecting Förster resonance energy transfer in living cells by conventional and spectral flow cytometry
Language English Country United States Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't, Research Support, N.I.H., Extramural
Grant support
CA16672
MD Anderson Cancer Center
S10 RR029552
NCRR NIH HHS - United States
PubMed
34128311
DOI
10.1002/cyto.a.24472
Knihovny.cz E-resources
- Keywords
- FRET, cell-based reporter assay, flow cytometry, kinase assay, protein kinase A, protein kinase B/AKT, spectral flow cytometry,
- MeSH
- Luminescent Proteins genetics metabolism MeSH
- Cyclic AMP-Dependent Protein Kinases metabolism MeSH
- Proto-Oncogene Proteins c-akt * metabolism MeSH
- Flow Cytometry methods MeSH
- Fluorescence Resonance Energy Transfer * methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
- Names of Substances
- Luminescent Proteins MeSH
- Cyclic AMP-Dependent Protein Kinases MeSH
- Proto-Oncogene Proteins c-akt * MeSH
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
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