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

Autor: Henderson J; Department of Lymphoma and Myeloma, The University of Texas-MD Anderson Cancer Center, Houston, Texas, USA., Havranek O; Department of Lymphoma and Myeloma, The University of Texas-MD Anderson Cancer Center, Houston, Texas, USA.; BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic.; Department of Hematology, Charles University and General University Hospital, Prague, Czech Republic., Ma MCJ; Department of Lymphoma and Myeloma, The University of Texas-MD Anderson Cancer Center, Houston, Texas, USA., Herman V; BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic.; Department of Hematology, Charles University and General University Hospital, Prague, Czech Republic., Kupcova K; BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic., Chrbolkova T; BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic., Pacheco-Blanco M; BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic., Wang Z; Department of Lymphoma and Myeloma, The University of Texas-MD Anderson Cancer Center, Houston, Texas, USA., Comer JM; Department of Lymphoma and Myeloma, The University of Texas-MD Anderson Cancer Center, Houston, Texas, USA., Zal T; Department of Leukemia, The University of Texas-MD Anderson Cancer Center, Houston, Texas, USA., Davis RE; 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.
Jazyk: angličtina
Zdroj: Cytometry. Part A : the journal of the International Society for Analytical Cytology [Cytometry A] 2022 Oct; Vol. 101 (10), pp. 818-834. Date of Electronic Publication: 2021 Jun 24.
DOI: 10.1002/cyto.a.24472
Abstrakt: 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.
(© 2021 International Society for Advancement of Cytometry.)
Databáze: MEDLINE
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