title: Measurement of Export to the Cytosol in Dendritic Cells Using a Cytofluorimetry-Based Assay.
abstract: The presentation of exogenous antigens on MHC class I molecules, known as cross-presentation, is a key function of dendritic cells (DCs). Cross-presentation via the cytosolic pathway involves antigen export from endocytic compartments to the cytosol. We have recently developed a cytofluorimetry-based assay to examine the kinetics and the efficiency of antigen export to the cytosol in DC populations. In this assay, DCs are loaded with a FRET-sensitive cytosolic substrate of β-lactamase, CCF4. Following uptake of β-lactamase by the DCs, the enzyme undergoes export to the cytosol leading to cleavage of the FRET dye. This cleavage and switch of fluorescence are analyzed by flow cytometry, allowing a quantitative measurement of this event.
authors.affiliation: INSERM U932 and Institut Curie, Centre de recherche, 26 Rue d'Ulm, 75005, Paris, France.
authors.forename: Omar I
authors.initials: OI
authors.lastname: Vivar
authors.affiliation: INSERM U932 and Institut Curie, Centre de recherche, 26 Rue d'Ulm, 75005, Paris, France. joao.magalhaes@curie.fr.
authors.forename: Joao G
authors.initials: JG
authors.lastname: Magalhaes
authors.affiliation: INSERM U932 and Institut Curie, Centre de recherche, 26 Rue d'Ulm, 75005, Paris, France.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Amigorena
mesh_terms: D000818:Animals; D017951:Antigen Presentation; D045142:Cross-Priming; D003600:Cytosol / Q000378:metabolism*; D003713:Dendritic Cells / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D051379:Mice; D021381:Protein Transport; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D001618:beta-Lactamases
keywords: CCF4; Cross-presentation; Cytosolic export; Dendritic cell; β-lactamase