title: Monitoring the intracellular routing of internalized antigens by immunofluorescence microscopy.
abstract: Professional antigen-presenting cells such as dendritic cells (DCs) and macrophages internalize extracellular antigens, process them intracellularly, and present the resulting antigen-derived peptides in the context of MHC I or MHC II molecules. Since the intracellular routing of the antigen determines whether antigens are presented on MHC I or MHC II molecules, a profound analysis of the intracellular distribution of the internalized antigens is of high interest. Here, we describe an immunofluorescence protocol to monitor the intracellular routing of the model-antigen Ovalbumin in bone marrow-derived dendritic cells (BM-DCs). This protocol describes a procedure to stain such cells with antibodies against different endosomal markers, such as EEA1 and LAMP1, and can be easily adopted to other antigen-presenting cells or antigens.
authors: Institutes of Molecular Medicine and Experimental Immunology (IMMEI), University Hospital Bonn, Bonn, Germany.
authors: Lars
authors: L
authors: Franken
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; D001854:Bone Marrow Cells / Q000166:cytology; D039521:CD11c Antigen / Q000378:metabolism; D002469:Cell Separation; D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism; D007150:Immunohistochemistry; D042541:Intracellular Space / Q000378:metabolism*; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D010047:Ovalbumin / Q000378:metabolism*; D021381:Protein Transport; D033921:Vesicular Transport Proteins / Q000378:metabolism*
chemical_list: D039521:CD11c Antigen; D052118:Lysosomal-Associated Membrane Protein 1; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D010047:Ovalbumin