title: Plasma Membrane Sheets for Studies of B Cell Antigen Internalization from Immune Synapses.
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.
authors.affiliation: Laboratory of Activation of Immune Receptors, Francis Crick Institute, Mill Hill Laboratory, London, UK.
authors.forename: Carla R
authors.initials: CR
authors.lastname: Nowosad
authors.affiliation: Laboratory of Activation of Immune Receptors, Francis Crick Institute, Mill Hill Laboratory, London, UK. pavel.tolar@crick.ac.uk.|Division of Immunology and Inflammation, Department of Medicine, Imperial College London, NW7 1AA, London, UK. pavel.tolar@crick.ac.uk.
authors.forename: Pavel
authors.initials: P
authors.lastname: Tolar
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000737:chemistry* / Q000276:immunology; D002462:Cell Membrane / Q000737:chemistry* / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D054992:Immunological Synapses / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D011947:Receptors, Antigen, B-Cell / Q000737:chemistry* / Q000276:immunology
chemical_list: D011947:Receptors, Antigen, B-Cell
keywords: Antigen internalization; B cell receptor; B cells; Cytoskeleton; Immune synapse; Plasma membrane sheets; Signaling; Transendocytosis; Trogocytosis