Monitoring Intracellular Routing of Internalized Antigens by Immunofluorescence Microscopy.

ABSTRACT

Antigen-presenting cells (APCs), especially macrophages and dendritic cells (DCs), are important for the induction of an adaptive immune response through their phagocytic capacity. APCs internalize extracellular antigens and, dependent on their intracellular localization, antigen-derived peptides are presented on MHC I or MHC II molecules. In context of antigen presentation and T cell activation tracking of internalized antigens is of high interest. In this article, we provide an immunofluorescence protocol and illustrate the analysis of intracellular routing of internalized antigens using the example of the model-antigen ovalbumin (OVA) in bone marrow-derived dendritic cells (BM-DCs). This protocol describes a procedure to stain such cells with an antibody against EEA-1, a marker for early endosomes, which can be easily adapted to other endosome markers, antigen-presenting cells, or antigens.