title: FRET Imaging by Laser Scanning Cytometry on Large Populations of Adherent Cells.
abstract: The application of FRET (fluorescence resonance energy transfer) sensors for monitoring protein-protein interactions under vital conditions is attracting increasing attention in molecular and cell biology. Laser-scanning cytometry (LSC), a slide-based sister procedure to flow cytometry, provides an opportunity to analyze large populations of adherent cells or 2-D solid tissues in their undisturbed physiological settings. Here we provide an LSC-based three-laser protocol for high-throughput ratiometric FRET measurements utilizing cyan and yellow fluorescent proteins as a FRET pair. Membrane labeling with Cy5 dye is used for cell identification and contouring. Pixel-by-pixel and single-cell FRET efficiencies are calculated to estimate the extent of the molecular interactions and their distribution in the cell populations examined. We also present a non-high-throughput donor photobleaching FRET application, for obtaining the required instrument parameters for ratiometric FRET. In the biological model presented, HeLa cells are transfected with the ECFP- or EYFP-tagged Fos and Jun nuclear proteins, which heterodimerize to form active AP1 transcription factor.
authors.affiliation: Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary; These authors contributed equally to this work.
authors.forename: Quang-Minh
authors.initials: QM
authors.lastname: Doan-Xuan
authors.forename: Nikoletta
authors.initials: N
authors.lastname: Szalóki
authors.forename: Katalin
authors.initials: K
authors.lastname: Tóth
authors.forename: János
authors.initials: J
authors.lastname: Szöllősi
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Bacso
authors.forename: György
authors.initials: G
authors.lastname: Vámosi
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002448:Cell Adhesion; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047569:Laser Scanning Cytometry / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; C527701:enhanced cyan fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins
keywords: ECFP-EYFP; donor photobleaching FRET; imaging cytometry; intensity based ratiometric FRET; laser-scanning cytometry; protein-protein interaction; slide-based cytometry