Abstract
Surrogate planar and membrane systems have been employed to study the architecture of immune synapses; however, they often do not recapitulate trans-synaptic extraction and endocytosis of ligands by the immune cells. Transendocytosis (or trogocytosis) of antigen from immune synapses is particularly critical for antigen processing and presentation by B cells. Here we describe a protocol for preparation of plasma membrane sheets (PMSs), which are flexible and fluid membrane substrates that support robust B cell antigen extraction. We show how to attach B cell antigens to the PMSs and how to investigate antigen extraction and endocytosis by fluorescent microscopy and computational image analysis. These techniques should be broadly applicable to studies of transendocytosis in a variety of cellular systems.
Key words
- B cells
- B cell receptor
- Immune synapse
- Signaling
- Cytoskeleton
- Antigen internalization
- Plasma membrane sheets
- Transendocytosis
- Trogocytosis
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Acknowledgements
This work was funded by the Francis Crick Institute, which receives its core funding from Cancer Research UK, the UK Medical Research Council and the Wellcome Trust. In addition, this work was supported by the European Research Council Consolidator Grant number 648228 and by the EMBO Young Investigator Programme.
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Nowosad, C.R., Tolar, P. (2017). Plasma Membrane Sheets for Studies of B Cell Antigen Internalization from Immune Synapses. In: Baldari, C., Dustin, M. (eds) The Immune Synapse. Methods in Molecular Biology, vol 1584. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6881-7_6
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DOI: https://doi.org/10.1007/978-1-4939-6881-7_6
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