Abstract
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.
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References
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.
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.
Pierce, S. K. (2002) Lipid rafts and B-cell activation, Nat Rev Immunol 2, 96–105.
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.
Axelrod, D. (1981) Cell-substrate contacts illuminated by total internal reflection fluorescence, J Cell Biol 89, 141–145.
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.
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.
Evans, J., and Yue, D. T. (2003) New turf for CFP/YFP FRET imaging of membrane signaling molecules, Neuron 38, 145–147.
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.
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.
Ciruela, F. (2008) Fluorescence-based methods in the study of protein-protein interactions in living cells, Curr Opin Biotechnol 19, 338–343.
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.
Zal, T., and Gascoigne, N. R. (2004) Photobleaching-corrected FRET efficiency imaging of live cells, Biophys J 86, 3923–3939.
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.
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.
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Sohn, H.W., Tolar, P., Brzostowski, J., Pierce, S.K. (2010). 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. In: Papkovsky, D. (eds) Live Cell Imaging. Methods in Molecular Biology, vol 591. Humana Press. https://doi.org/10.1007/978-1-60761-404-3_10
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DOI: https://doi.org/10.1007/978-1-60761-404-3_10
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