Monitoring the intracellular routing of internalized antigens by immunofluorescence microscopy

Methods Mol Biol. 2013:960:371-377. doi: 10.1007/978-1-62703-218-6_27.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bone Marrow Cells / cytology
  • CD11c Antigen / metabolism
  • Cell Separation
  • Dendritic Cells / cytology
  • Dendritic Cells / metabolism
  • Immunohistochemistry
  • Intracellular Space / metabolism*
  • Lysosomal-Associated Membrane Protein 1 / metabolism*
  • Mice
  • Microscopy, Fluorescence / methods*
  • Ovalbumin / metabolism*
  • Protein Transport
  • Vesicular Transport Proteins / metabolism*

Substances

  • CD11c Antigen
  • Lysosomal-Associated Membrane Protein 1
  • Vesicular Transport Proteins
  • early endosome antigen 1
  • Ovalbumin