High throughput FRET analysis of protein–protein interactions by slide-based imaging laser scanning cytometry

Authors

  • Nikoletta Szalóki,

    1. Department of Biophysics and Cell Biology, Medical and Health Science Center, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary
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  • Quang Minh Doan-Xuan,

    1. Department of Biophysics and Cell Biology, Medical and Health Science Center, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary
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  • János Szöllősi,

    1. Department of Biophysics and Cell Biology, Medical and Health Science Center, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary
    2. HASc-UD Cell Biology and Signaling Research Group, Medical and Health Science Center, University of Debrecen, Hungary
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  • Katalin Tóth,

    1. German Cancer Research Center (DKFZ), Biophysics of Macromolecules (B040), Heidelberg, Germany
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  • György Vámosi,

    Corresponding author
    • Department of Biophysics and Cell Biology, Medical and Health Science Center, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary
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  • Zsolt Bacsó

    Corresponding author
    • Department of Biophysics and Cell Biology, Medical and Health Science Center, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary
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  • This article is dedicated to the memory of Dr. Robert Clegg, a pioneer and leading expert in FRET techniques.

  • Nikoletta Szalóki and Quang Minh Doan-Xuan are equal first authors.

  • György Vámosi and Zsolt Bacsó are equal senior authors.

Correspondence to: György Vámosi, Department of Biophysics and Cell Biology, Medical and Health Science Center, Research Center for Molecular Medicine, University of Debrecen, 98 Nagyerdei krt., H-4032 Debrecen, Hungary. E-mail: vamosig@med.unideb.hu and Zsolt Bacsó, Department of Biophysics and Cell Biology, Medical and Health Science Center, Research Center for Molecular Medicine, University of Debrecen, 98 Nagyerdei krt., H-4032 Debrecen, Hungary. E-mail: bacso@med.unideb.hu

Abstract

Laser scanning cytometry (LSC) is a slide-based technique combining advantages of flow and image cytometry: automated, high-throughput detection of optical signals with subcellular resolution. Fluorescence resonance energy transfer (FRET) is a spectroscopic method often used for studying molecular interactions and molecular distances. FRET has been measured by various microscopic and flow cytometric techniques. We have developed a protocol for a commercial LSC instrument to measure FRET on a cell-by-cell or pixel-by-pixel basis on large cell populations, which adds a new modality to the use of LSC. As a reference sample for FRET, we used a fusion protein of a single donor and acceptor (ECFP-EYFP connected by a seven-amino acid linker) expressed in HeLa cells. The FRET efficiency of this sample was determined via acceptor photobleaching and used as a reference value for ratiometric FRET measurements. Using this standard allowed the precise determination of an important parameter (the alpha factor, characterizing the relative signal strengths from a single donor and acceptor molecule), which is indispensable for quantitative FRET calculations in real samples expressing donor and acceptor molecules at variable ratios. We worked out a protocol for the identification of adherent, healthy, double-positive cells based on light-loss and fluorescence parameters, and applied ratiometric FRET equations to calculate FRET efficiencies in a semi-automated fashion. To test our protocol, we measured the FRET efficiency between Fos-ECFP and Jun-EYFP transcription factors by LSC, as well as by confocal microscopy and flow cytometry, all yielding nearly identical results. Our procedure allows for accurate FRET measurements and can be applied to the fast screening of protein interactions. A pipeline exemplifying the gating and FRET analysis procedure using the CellProfiler software has been made accessible at our web site. © 2013 International Society for Advancement of Cytometry

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