title: Analysis of Phagosomal Antigen Degradation by Flow Organellocytometry.
abstract: Professional phagocytes internalize self and non-self particles by phagocytosis to initiate innate immune responses. After internalization, the formed phagosome matures through fusion and fission events with endosomes and lysosomes to obtain a more acidic, oxidative and hydrolytic environment for the degradation of its cargo. Interestingly, phagosome maturation kinetics differ between cell types and cell activation states. This protocol allows to quantify phagosome maturation kinetics on a single organelle level in different types of phagocytes using flow cytometry. Here, ovalbumin (OVA)-coupled particles are used as phagocytosis model system in dendritic cells (DC), which are internalized by phagocytosis. After different time points, phagosome maturation parameters, such as phagosomal degradation of OVA and acquisition of lysosomal proteins (like LAMP-1), can be measured simultaneously in a highly quantitative manner by flow organellocytometry. These read-outs can be correlated to other phagosomal functions, for example antigen degradation, processing and loading in DC.
authors.affiliation: Institute Curie, INSERM U932, PSL Research University, Paris, France; VIB Inflammation Research Center, Unit of Molecular Signal Transduction in Inflammation, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors.forename: Eik
authors.initials: E
authors.lastname: Hoffmann
authors.affiliation: VIB Inflammation Research Center, Unit of Molecular Signal Transduction in Inflammation, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Pauwels
authors.affiliation: Institute Curie, INSERM U932, PSL Research University, Paris, France.
authors.forename: Andrés
authors.initials: A
authors.lastname: Alloatti
authors.affiliation: Institute Curie, INSERM U932, PSL Research University, Paris, France; Department of Virology, University of Buenos Aires, Buenos Aires, Argentina.
authors.forename: Fiorella
authors.initials: F
authors.lastname: Kotsias
authors.affiliation: Institute Curie, INSERM U932, PSL Research University, Paris, France.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Amigorena