Detecting Förster Resonance Energy Transfer in Living Cells by Conventional and Spectral Flow Cytometry

Authors

Jared Henderson
Ondrej Havranek
Man Chun John Ma
Vaclav Herman
Kristyna Kupcova
Tereza Chrbolkova
Mariana Pacheco-Blanco
Zhiqiang Wang
Justin M Comer
Tomasz Zal
Richard Eric Davis

Publication Date

10-1-2022

Journal

Cytometry A.

DOI

10.1002/cyto.a.24472

PMID

34128311

PMCID

PMC12175677

PubMedCentral® Posted Date

6-18-2025

PubMedCentral® Full Text Version

Author MSS

Abstract

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.

Keywords

Cyclic AMP-Dependent Protein Kinases, Flow Cytometry, Fluorescence Resonance Energy Transfer, Luminescent Proteins, Proto-Oncogene Proteins c-akt, Protein kinase B/AKT, Protein kinase A, cell-based reporter assay, FRET, kinase assay, flow cytometry, spectral flow cytometry

Published Open-Access

yes

Recommended Citation

Henderson, Jared; Havranek, Ondrej; Ma, Man Chun John; et al., "Detecting Förster Resonance Energy Transfer in Living Cells by Conventional and Spectral Flow Cytometry" (2022). Faculty, Staff and Student Publications. 4336.
https://digitalcommons.library.tmc.edu/uthgsbs_docs/4336