Chapter title |
Imaging the Interactions Between B Cells and Antigen-Presenting Cells
|
---|---|
Chapter number | 10 |
Book title |
B Cell Receptor Signaling
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7474-0_10 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7473-3, 978-1-4939-7474-0
|
Authors |
Jia C. Wang, Madison Bolger-Munro, Michael R. Gold |
Abstract |
In vivo, B cells are often activated by antigens that are displayed on the surface of antigen-presenting cells (APCs). Binding of membrane-associated antigens to the B cell receptor (BCR) causes rapid cytoskeleton-dependent changes in the spatial organization of the BCR and other B cell membrane proteins, leading to the formation of an immune synapse. This process has been modeled using antigens attached to artificial planar lipid bilayers or to plasma membrane sheets. As a more physiological system for studying B cell-APC interactions, we have expressed model antigens in easily transfected adherent cell lines such as Cos-7 cells. The model antigens that we have used are a transmembrane form of a single-chain anti-Igκ antibody and a transmembrane form of hen egg lysozyme that is fused to a fluorescent protein. This has allowed us to study multiple aspects of B cell immune synapse formation including cytoskeletal reorganization, BCR microcluster coalescence, BCR-mediated antigen gathering, and BCR signaling. Here, we provide protocols for expressing these model antigens on the surface of Cos-7 cells, transfecting B cells with siRNAs or with plasmids encoding fluorescent proteins, using fixed cell and live cell fluorescence microscopy to image B cell-APC interactions, and quantifying APC-induced changes in BCR spatial organization and signaling. |
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