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

  • 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,
  • 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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Literature Cited

Literature Cited
  Bacso, Z. and Eliason, J.F. 2001. Measurement of DNA damage associated with apoptosis by laser scanning cytometry. Cytometry 45:180‐186.
  Bacso, Z., Bene, L., Bodnar, A., Matko, J., and Damjanovich, S. 1996. A photobleaching energy transfer analysis of CD8/MHC‐I and LFA‐1/ICAM‐1 interactions in CTL‐target cell conjugates. Immunol. Lett. 54:151‐156.
  Bacso, Z., Everson, R.B., and Eliason, J.F. 2000. The DNA of annexin V‐binding apoptotic cells is highly fragmented. Cancer Res. 60:4623‐4628.
  Bacso, Z., Bene, L., Damjanovich, L., and Damjanovich, S. 2002. INF‐gamma rearranges membrane topography of MHC‐I and ICAM‐1 in colon carcinoma cells. Biochem. Biophys. Res. Comm. 290:635‐640.
  Baudendistel, N., Muller, G., Waldeck, W., Angel, P., and Langowski, J. 2005. Two‐hybrid fluorescence cross‐correlation spectroscopy detects protein‐protein interactions in vivo. Chemphyschem 6:984‐990.
  Carpenter, A.E., Jones, T.R., Lamprecht, M.R., Clarke, C., Kang, I.H., Friman, O., Guertin, D.A., Chang, J.H., Lindquist, R.A., Moffat, J., Golland, P., and Sabatini, D.M. 2006. CellProfiler: Image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7:R100.
  Clatch, R.J. 2001. Immunophenotyping of hematological malignancies by laser scanning cytometry. Methods Cell Biol. 64:313‐342.
  Clatch, R.J., Walloch, J.L., Zutter, M.M., and Kamentsky, L.A. 1996. Immunophenotypic analysis of hematologic malignancy by laser scanning cytometry. Am. J. Clin. Pathol. 105:744‐755.
  Clegg, R. 2009. Förster resonance energy transfer—FRET: What is it, why do it, and how it's done. In FRET and FLIM Techniques. Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 33 (T.W.J. Gadella, ed.) pp. 1‐57. Elsevier, Amsterdam.
  Damjanovich, S., Tron, L., Szöllősi, J., Zidovetzki, R., Vaz, W.L., Regateiro, F., Arndt‐Jovin, D.J., and Jovin, T.M. 1983. Distribution and mobility of murine histocompatibility H‐2Kk antigen in the cytoplasmic membrane. Proc. Natl. Acad. Sci. U.S.A. 80:5985‐5989.
  Darzynkiewicz, Z. and Bedner, E. 2000. Analysis of apoptotic cells by flow and laser scanning cytometry. Methods Enzymol. 322:18‐39.
  Gorczyca, W., Darzynkiewicz, Z., and Melamed, M.R. 1997. Laser scanning cytometry in pathology of solid tumors. A review. Acta Cytolog. 41:98‐108.
  Gorczyca, W., Deptala, A., Bedner, E., Li, X., Melamed, M.R., and Darzynkiewicz, Z. 2001. Analysis of human tumors by laser scanning cytometry. Methods Cell Biol. 64:421‐443.
  Harnett, M.M. 2007. Laser scanning cytometry: Understanding the immune system in situ. Nat. Rev. Immunol. 7:897‐904.
  Henriksen, M., Miller, B., Newmark, J., Al‐Kofahi, Y., and Holden, E. 2011. Laser scanning cytometry and its applications: A pioneering technology in the field of quantitative imaging cytometry. Methods Cell Biol. 102:161‐205.
  Horvath, G., Petras, M., Szentesi, G., Fabian, A., Park, J.W., Vereb, G., and Szöllősi, J. 2005. Selecting the right fluorophores and flow cytometer for fluorescence resonance energy transfer measurements. Cytometry A 65:148‐157.
  Imamura, H., Nhat, K.P., Togawa, H., Saito, K., Iino, R., Kato‐Yamada, Y., Nagai, T., and Noji, H. 2009. Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer‐based genetically encoded indicators. Proc. Natl. Acad. Sci. U.S.A. 106:15651‐15656.
  Jovin, T.M. and Arndt‐Jovin, D.J. 1989. Luminescence digital imaging microscopy. Annu. Rev. Biophys. Biophys. Chem. 18:271‐308.
  Kamentsky, L.A. and Kamentsky, L.D. 1991. Microscope‐based multiparameter laser scanning cytometer yielding data comparable to flow cytometry data. Cytometry 12:381‐387.
  Luther, E., Kamentsky, L., Henriksen, M., and Holden, E. 2004. Next‐generation laser scanning cytometry. Methods Cell Biol. 75:185‐218.
  Malkani, N. and Schmid, J.A. 2011. Some secrets of fluorescent proteins: Distinct bleaching in various mounting fluids and photoactivation of cyan fluorescent proteins at YFP‐excitation. PloS One 6:e18586.
  Nagy, P., Bene, L., Hyun, W.C., Vereb, G., Braun, M., Antz, C., Paysan, J., Damjanovich, S., Park, J.W., and Szöllősi, J. 2005. Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins. Cytometry A 67:86‐96.
  Okumoto, S., Jones, A., and Frommer, W.B. 2012. Quantitative imaging with fluorescent biosensors. Annu. Rev. Plant Biol. 63:663‐706.
  Pollok, B.A. and Heim, R. 1999. Using GFP in FRET‐based applications. Trends Cell Biol. 9:57‐60.
  Poulsen, C.P., Vereb, G., Geshi, N., and Schulz, A. 2013. Inhibition of cytoplasmic streaming by cytochalasin D is superior to paraformaldehyde fixation for measuring FRET between fluorescent protein‐tagged Golgi components. Cytometry A 83:830‐838.
  Pozarowski, P., Holden, E., and Darzynkiewicz, Z. 2013. Laser scanning cytometry: Principles and applications‐an update. Methods Mol. Biol. 931:187‐212.
  Sebestyen, Z., Nagy, P., Horvath, G., Vámosi, G., Debets, R., Gratama, J.W., Alexander, D.R., and Szöllősi, J. 2002. Long wavelength fluorophores and cell‐by‐cell correction for autofluorescence significantly improves the accuracy of flow cytometric energy transfer measurements on a dual‐laser benchtop flow cytometer. Cytometry 48:124‐135.
  Szabo, G. Jr., Pine, P.S., Weaver, J.L., Kasari, M., and Aszalos, A. 1992. Epitope mapping by photobleaching fluorescence resonance energy transfer measurements using a laser scanning microscope system. Biophys. J. 61:661‐670.
  Szalóki, N., Doan‐Xuan, Q.M., Szöllősi, J., Toth, K., Vámosi, G., and Bacso, Z. 2013. High throughput FRET analysis of protein‐protein interactions by slide‐based imaging laser scanning cytometry. Cytometry A 83:818‐829.
  Szentesi, G., Vereb, G., Horvath, G., Bodnar, A., Fabian, A., Matko, J., Gaspar, R., Damjanovich, S., Matyus, L., and Jenei, A. 2005. Computer program for analyzing donor photobleaching FRET image series. Cytometry A 67:119‐128.
  Szöllősi, J., Tron, L., Damjanovich, S., Helliwell, S.H., Arndt‐Jovin, D., and Jovin, T.M. 1984. Fluorescence energy transfer measurements on cell surfaces: A critical comparison of steady‐state fluorimetric and flow cytometric methods. Cytometry 5:210‐216.
  Tron, L., Szöllősi, J., Damjanovich, S., Helliwell, S.H., Arndt‐Jovin, D.J., and Jovin, T.M. 1984. Flow cytometric measurement of fluorescence resonance energy transfer on cell surfaces. Quantitative evaluation of the transfer efficiency on a cell‐by‐cell basis. Biophys. J. 45:939‐946.
  Vámosi, G., Bodnar, A., Vereb, G., Jenei, A., Goldman, C.K., Langowski, J., Toth, K., Matyus, L., Szöllősi, J., Waldmann, T.A., and Damjanovich, S. 2004. IL‐2 and IL‐15 receptor alpha‐subunits are coexpressed in a supramolecular receptor cluster in lipid rafts of T cells. Proc. Natl. Acad. Sci. U.S.A. 101:11082‐11087.
  Vámosi, G., Baudendistel, N., von der Lieth, C.W., Szalóki, N., Mocsar, G., Muller, G., Brazda, P., Waldeck, W., Damjanovich, S., Langowski, J., and Toth, K. 2008a. Conformation of the c‐Fos/c‐Jun complex in vivo: A combined FRET, FCCS, and MD‐modeling study. Biophys. J. 94:2859‐2868.
  Vámosi, G., Damjanovich, S., and Szöllősi, J. 2008b. Dissecting interacting molecular populations by FRET. Cytometry A 73:681‐684.
  Vereb, G., Nagy, P., and Szöllősi, J. 2011. Flow cytometric FRET analysis of protein interaction. Methods Mol. Biol. 699:371‐392.
  Woehler, A., Wlodarczyk, J., and Neher, E. 2010. Signal/noise analysis of FRET‐based sensors. Biophys. J. 99:2344‐2354.
Internet Resources
  Download site for CellProfiler cell image analysis software.
  Online curve‐fitting and surface‐fitting Web site.
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