Live Cell Imaging pp 159-183

Part of the Methods in Molecular Biology book series (MIMB, volume 591)

A Method for Analyzing Protein–Protein Interactions in the Plasma Membrane of Live B Cells by Fluorescence Resonance Energy Transfer Imaging as Acquired by Total Internal Reflection Fluorescence Microscopy

  • Hae Won Sohn
  • Pavel Tolar
  • Joseph Brzostowski
  • Susan K. Pierce


For more than a decade, fluorescence resonance energy transfer (FRET) imaging methods have been developed to study dynamic interactions between molecules at the nanometer scale in live cells. Here, we describe a protocol to measure FRET by the acceptor-sensitized emission method as detected by total internal reflection fluorescence (TIRF) imaging to study the interaction of appropriately labeled plasma membrane-associated molecules that regulate the earliest stages of antigen-mediated signaling in live B lymphocytes. This protocol can be adapted and applied to many cell types where there is an interest in understanding signal transduction mechanisms in live cells.

Key words

B lymphocyte fluorescence resonance energy transfer (FRET) total internal reflection fluorescence (TIRF) imaging B cell receptor (BCR) signaling planar lipid bilayer immune synapse 


  1. 1.
    Fearon, D. T., and Carroll, M. C. (2000) Regulation of B lymphocyte responses to foreign and self-antigens by the CD19/CD21 complex, Annu Rev Immunol 18, 393–422.CrossRefPubMedGoogle Scholar
  2. 2.
    Dal Porto, J. M., Gauld, S. B., Merrell, K. T., Mills, D., Pugh-Bernard, A. E., and Cambier, J. (2004) B cell antigen receptor signaling 101, Mol Immunol 41, 599–613.CrossRefPubMedGoogle Scholar
  3. 3.
    Pierce, S. K. (2002) Lipid rafts and B-cell activation, Nat Rev Immunol 2, 96–105.CrossRefPubMedGoogle Scholar
  4. 4.
    Tolar, P., Sohn, H. W., and Pierce, S. K. (2008) Viewing the antigen-induced initiation of B-cell activation in living cells, Immunol Rev 221, 64–76.CrossRefPubMedGoogle Scholar
  5. 5.
    Axelrod, D. (1981) Cell-substrate contacts illuminated by total internal reflection fluorescence, J Cell Biol 89, 141–145.CrossRefPubMedGoogle Scholar
  6. 6.
    Kandere-Grzybowska, K., Campbell, C., Komarova, Y., Grzybowski, B. A., and Borisy, G. G. (2005) Molecular dynamics imaging in micropatterned living cells, Nat Methods 2, 739–741.CrossRefPubMedGoogle Scholar
  7. 7.
    Brian, A. A., and McConnell, H. M. (1984) Allogeneic stimulation of cytotoxic T cells by supported planar membranes, Proc Natl Acad Sci U S A 81, 6159–6163.CrossRefPubMedGoogle Scholar
  8. 8.
    Evans, J., and Yue, D. T. (2003) New turf for CFP/YFP FRET imaging of membrane signaling molecules, Neuron 38, 145–147.CrossRefPubMedGoogle Scholar
  9. 9.
    Sohn, H. W., Tolar, P., and Pierce, S. K. (2008) Membrane heterogeneities in the formation of B cell receptor-Lyn kinase microclusters and the immune synapse, J Cell Biol 182, 367–379.CrossRefPubMedGoogle Scholar
  10. 10.
    Truong, K., and Ikura, M. (2001) The use of FRET imaging microscopy to detect protein-protein interactions and protein conformational changes in vivo, Curr Opin Struct Biol 11, 573–578.CrossRefPubMedGoogle Scholar
  11. 11.
    Ciruela, F. (2008) Fluorescence-based methods in the study of protein-protein interactions in living cells, Curr Opin Biotechnol 19, 338–343.CrossRefPubMedGoogle Scholar
  12. 12.
    van Rheenen, J., Langeslag, M., and Jalink, K. (2004) Correcting confocal acquisition to optimize imaging of fluorescence resonance energy transfer by sensitized emission, Biophys J 86, 2517–2529.CrossRefPubMedGoogle Scholar
  13. 13.
    Zal, T., and Gascoigne, N. R. (2004) Photobleaching-corrected FRET efficiency imaging of live cells, Biophys J 86, 3923–3939.CrossRefPubMedGoogle Scholar
  14. 14.
    Tolar, P., Sohn, H. W., and Pierce, S. K. (2005) The initiation of antigen-induced B cell antigen receptor signaling viewed in living cells by fluorescence resonance energy transfer, Nat Immunol 6, 1168–1176.CrossRefPubMedGoogle Scholar
  15. 15.
    Sohn, H. W., Tolar, P., Jin, T., and Pierce, S. K. (2006) Fluorescence resonance energy transfer in living cells reveals dynamic membrane changes in the initiation of B cell signaling, Proc Natl Acad Sci USA 103, 8143–8148.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Hae Won Sohn
    • 1
  • Pavel Tolar
    • 1
  • Joseph Brzostowski
    • 1
  • Susan K. Pierce
    • 1
  1. 1.Laboratory of Immunogenetics, National Institute of Allergy and Infectious DiseasesNational Institutes of HealthRockvilleUSA

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