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.
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Acknowledgments
This work was supported by the German Research Foundation (DFG) Grant SFB645 Project C1.
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Chasan, A.I., Beyer, M., Kurts, C., Burgdorf, S. (2013). Isolation of a Specialized, Antigen-Loaded Early Endosomal Subpopulation by Flow Cytometry. In: van Endert, P. (eds) Antigen Processing. Methods in Molecular Biology™, vol 960. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-218-6_28
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DOI: https://doi.org/10.1007/978-1-62703-218-6_28
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-217-9
Online ISBN: 978-1-62703-218-6
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