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Título: | Visualization of TCR Nanoclusters via Immunogold Labeling, Freeze-Etching, and Surface Replication |
Autor: | Fiala, Gina J.; Rejas, M. Teresa CSIC ORCID ; Schamel, Wolfgang; Santen, Hisse M. van | Palabras clave: | Receptor clustering T cell receptor Transmission electron microscopy Immunogold labeling Cell surface replicas Cell surface proteins |
Fecha de publicación: | 2013 | Editor: | Academic Press | Citación: | Methods in Cell Biology 117: 391- 410 (2013) | Resumen: | T cells show high sensitivity for antigen, even though their T-cell antigen receptor (TCR) has a low affinity for its ligand, a major histocompatibility complex molecule presenting a short pathogen-derived peptide. Over the past few years, it has become clear that these paradoxical properties rely at least in part on the organization of cell surface-expressed TCRs in TCR nanoclusters. We describe a protocol, comprising immunogold labeling, cell surface replica generation, and electron microscopy (EM) analysis that allows nanoscale resolution of the distribution of TCRs and other cell surface molecules of cells grown in suspension. Unlike most of the light microscopy-based single-molecule resolution techniques, this technique permits visualization of these molecules on cell surfaces that do not adhere to an experimental support. Given the potential of adhesion-induced receptor redistributions, our technique is a relevant complement to the substrate adherence-dependent techniques. Furthermore, it does not rely on introduction of fluorescently labeled recombinant molecules and therefore allows direct analysis of nonmanipulated primary cells. © 2013 Elsevier Inc. | URI: | http://hdl.handle.net/10261/99624 | DOI: | 10.1016/B978-0-12-408143-7.00021-9 | Identificadores: | doi: 10.1016/B978-0-12-408143-7.00021-9 issn: 0091-679X |
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