FRET Imaging by Laser Scanning Cytometry on Large Populations of Adherent Cells

Quang‐Minh Doan‐Xuan1, Nikoletta Szalóki1, Katalin Tóth2, János Szöllősi3, Zsolt Bacso1, György Vámosi1

1 These authors contributed equally to this work, 2 German Cancer Research Center, Biophysics of Macromolecules, Heidelberg, 3 MTA‐DE Cell Biology and Signaling Research Group, University of Debrecen, Debrecen
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 2.23
DOI:  10.1002/0471142956.cy0223s70
Online Posting Date:  October, 2014
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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. Curr. Protoc. Cytom. 70:2.23.1‐2.23.29. © 2014 by John Wiley & Sons, Inc.

Keywords: laser‐scanning cytometry; slide‐based cytometry; imaging cytometry; intensity based ratiometric FRET; donor photobleaching FRET; protein‐protein interaction; ECFP‐EYFP

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Ratiometric FRET Measurement for Live Attached Cells by Laser‐Scanning Cytometry
  • Support Protocol 1: Donor Photobleaching FRET Measurement Applying Laser‐Scanning Cytometry
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Ratiometric FRET Measurement for Live Attached Cells by Laser‐Scanning Cytometry

  Materials
  • Plasmids:
    • Controls: pSV‐ECFP‐EYFP fusion; pSV‐ECFP; pSV‐EYFP
    • FRET samples: ECFP‐ and EYFP‐tagged proteins of interests, example in this protocol: pSV‐c‐Fos‐ECFP and pSV‐c‐Jun‐EYFP (Baudendistel et al., ; Szalóki et al., )
  • HeLa adherent cells
  • Cell culture medium for HeLa cells (see recipe)
  • Serum‐free RPMI medium
  • FuGeneHD transfection reagent (Promega)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Cy5 succinimidyl ester working solution (see recipe)
  • Hanks’ balanced salt solution (HBSS; see recipe)
  • Chambered coverslips, in our case μ‐Slide 8‐well chambered coverslip (ibiTreat, 180 μm bottom thickness, ibidi GmbH, http://ibidi.com)
  • iCys Research Imaging Cytometer (Thorlabs Imaging Systems) with:
    • Olympus IX‐71 inverted microscope
    • 40× air objective (0.75 N.A., Olympus)
    • 405‐nm, 488‐nm, and 633‐nm solid‐state lasers
    • Photodiode (forward scatter) detector (PD)
    • Three photomultiplier tubes (PMTs) with three filters in front: 460‐500 nm band‐pass filter, 535‐565 nm band‐pass filter, and 650‐nm long‐pass filter
    • High‐resolution scan‐step module
    • Autofocus controller
    • Motorized X‐Y stage and controller (Prior Scientific)
    • Remote control unit
    • Focus monitor
    • iCys 7.0 software
    • iCys specimen carriers, specified for chambered coverslips
  • Emission filter: 460‐ to 500‐nm band‐pass for the donor channel ECFP (AHF Analysentechnik)
  • DP71 color camera (Olympus Hungary)
  • CellProfiler 2.1.0 open‐source image‐processing software (Carpenter et al., )

Support Protocol 1: Donor Photobleaching FRET Measurement Applying Laser‐Scanning Cytometry

  Additional Materials protocol 1Basic Protocol
  • 20× air objective (0.5 N.A., Olympus) and a 60× water immersion objective (1.2 N.A., Olympus) are needed
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Figures

Videos

Literature Cited

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Internet Resources
  http://www.cellprofiler.org/
  Download site for CellProfiler cell image analysis software.
  http://zunzun.com/
  Online curve‐fitting and surface‐fitting Web site.
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