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Detecting Förster resonance energy transfer in living cells by conventional and spectral flow cytometry
J. Henderson, O. Havranek, MCJ. Ma, V. Herman, K. Kupcova, T. Chrbolkova, M. Pacheco-Blanco, Z. Wang, JM. Comer, T. Zal, RE. Davis
Language English Country United States
Document type Journal Article
Grant support
RR029552
NCI NIH HHS - United States
S10
NCI NIH HHS - United States
RR029552
NCI NIH HHS - United States
S10
NCI NIH HHS - United States
NLK
Free Medical Journals
from 2003 to 1 year ago
Medline Complete (EBSCOhost)
from 2012-06-01 to 1 year ago
Wiley Free Content
from 2003 to 1 year ago
PubMed
34128311
DOI
10.1002/cyto.a.24472
Knihovny.cz E-resources
- 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
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
References provided by Crossref.org
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