title: Preparation of intact, highly purified phagosomes from Dictyostelium.
abstract: Phagocytosis plays a fundamental role in the immune system for the defense against invading microorganisms and the clearing of apoptotic and cancerous cells. The common amoeba Dictyostelium discoideum is a recognized model for professional immune phagocytes and is now commonly used to study host-pathogen interactions. Dictyostelium is genetically and biochemically tractable and is a most versatile experimental system. The classical protocol for purifying phagosomes formed by ingestion of latex beads particles has been adapted to Dictyostelium. It was improved in yield, purity, and synchronicity, allowing isolation of milligram amounts of phagosomal proteins and lipids. This method has been used successfully to highlight membrane trafficking and phagosome maturation. Here, we present a step-by-step protocol including detailed notes necessary for ensuring access to a large number of highly synchronized phagosomes of high purity and integrity.
authors.affiliation: Department of Molecular Cell Research, Max-Planck-Institute for Medical Research, Heidelberg, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Gotthardt
authors.forename: Régis
authors.initials: R
authors.lastname: Dieckmann
authors.forename: Vincent
authors.initials: V
authors.lastname: Blancheteau
authors.forename: Claudia
authors.initials: C
authors.lastname: Kistler
authors.forename: Frank
authors.initials: F
authors.lastname: Reichardt
authors.forename: Thierry
authors.initials: T
authors.lastname: Soldati
mesh_terms: D000818:Animals; D004023:Dictyostelium / Q000166:cytology / Q000378:metabolism*; D007840:Latex; D008863:Microspheres; D010588:Phagosomes / Q000737:chemistry*
chemical_list: D007840:Latex