Abstract
Extensive studies have been performed in order to understand the interaction of receptors with soluble ligands. However, we know very little of the parameters that regulate the interaction of receptors with membrane-bound ligands. Artificial lipid bilayers can be used to mimic cell-to-cell interactions, but a major challenge remains how to tether molecules to these membranes. We describe a simple and reliable method to tether ligands on glass-supported artificial bilayers containing biotinylated lipids. In this system, the model antigen hen egg lysozyme (HEL) is tethered through a fluorescently labeled streptavidin-monobiotinylated anti-HEL antibody bridge. This allows us to study the interaction of HEL-specific B cells with the tethered antigen by a variety of microscopy techniques. We recently used this system to study the activation of B cells by membrane antigens.
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References
Hladky SB, Haydon DA. Discreteness of conductance change in bimolecular lipid membranes in the presence of certain antibiotics. Nature 1970;225:451–3.
Mueller P, Rudin DO, Tien HT, Wescott WC. Reconstitution of cell membrane structure in vitro and its transformation into an excitable system. Nature 1962;194:979–80.
Hafeman DG, von Tscharner V, McConnell HM. Specific antibody-dependent interactions between macrophages and lipid haptens in planar lipid monolayers. Proc Natl Acad Sci USA 1981;78:4552–6.
Brian AA, McConnell HM. Allogeneic stimulation of cytotoxic T cells by supported planar membranes. Proc Natl Acad Sci USA 1984;81:6159–63.
Dustin ML, Ferguson LM, Chan PY, Springer TA, Golan DE. Visualization of CD2 interaction with LFA-3 and determination of the two-dimensional dissociation constant for adhesion receptors in a contact area. J Cell Biol 1996;132:465–74.
Grakoui A, Bromley SK, Sumen C, Davis MM, Shaw AS, Allen PM, Dustin ML. The immunological synapse: A molecular machine controlling T cell activation. Science 1999;285:221–7.
Groves JT, Dustin ML. Supported planar bilayers in studies on immune cell adhesion and communication. J Immunol Methods 2003;278:19–32.
Carrasco YR, Batista FD. B-cell activation by membrane-bound antigens is facilitated by the interaction of VLA-4 with VCAM-1. EMBO J 2006;25:889–99.
Carrasco YR, Fleire SJ, Cameron T, Dustin ML, Batista FD. LFA-1/ICAM-1 interaction lowers the threshold of B cell activation by facilitating B cell adhesion and synapse formation. Immunity 2004;20:589–99.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Fleire, S.J., Batista, F.D. (2009). Studying Cell-to-Cell Interactions: An Easy Method of Tethering Ligands on Artificial Membranes. In: Larijani, B., Woscholski, R., Rosser, C. (eds) Lipid Signaling Protocols. Methods in Molecular Biology, vol 462. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-115-8_9
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DOI: https://doi.org/10.1007/978-1-60327-115-8_9
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-60327-115-8
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