title: Isolation of a specialized, antigen-loaded early endosomal subpopulation by flow cytometry.
abstract: Isolation and characterization of antigen-containing endosomes remains difficult utilizing standard purification techniques. Here, we describe a method, which allows isolation of antigen-loaded endosomes, that is based on flow cytometrical analysis and sorting. We specifically isolated antigen-containing endosomes from cells that had taken up fluorochrome-labeled ovalbumin via mannose receptor-mediated endocytosis. The protocol described here allows for the isolation of pure fractions of ovalbumin-containing endosomes and the extraction of proteins from these endosomes for analysis by western blot. Importantly, this protocol can be easily extended to other fluorochrome-labeled antigens and to primary antigen-presenting cells like bone marrow-derived dendritic cells.
authors: Life and Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany.
authors: Achmet Imam
authors: AI
authors: Chasan
authors: Life and Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany.
authors: Marc
authors: M
authors: Beyer
authors: Institutes of Molecular Medicine and Experimental Immunology (IMMEI), University Hospital Bonn, Bonn, Germany.
authors: Christian
authors: C
authors: Kurts
authors: Life and Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany. burgdorf@uni-bonn.de.
authors: Sven
authors: S
authors: Burgdorf
mesh_terms: D000818:Animals; D000938:Antigen-Presenting Cells / Q000166:cytology; D011992:Endosomes / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D037181:Lectins, C-Type / Q000235:genetics; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins / Q000235:genetics; D051379:Mice; D010047:Ovalbumin / Q000302:isolation & purification / Q000378:metabolism*; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000235:genetics; D014162:Transfection
chemical_list: D005456:Fluorescent Dyes; D037181:Lectins, C-Type; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D011956:Receptors, Cell Surface; D010047:Ovalbumin