title: Analysis of Intracellular Trafficking of Dendritic Cell Receptors for Antigen Targeting.
abstract: Antibody-targeted vaccination aims to efficiently deliver antigen to dendritic cells by targeting specific receptors at the cell surface. The choice of receptor depends on different factors, including their capacity to induce internalization of the delivered antigen/adjuvant cargo. Assays currently used to monitor internalization in dendritic cells have several limitations. We have developed a novel DNA-based probe that allows for simple and robust high-throughput analysis of internalization. Designed for flow cytometry, the probe can also be used for fluorescence microscopy to clearly distinguish internalized from surface-bound material. Here, we describe the steps for modifying material (antibodies, proteins) with the probe, undertaking the assay, and analyzing the data obtained from flow cytometry.
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Rd, Parkville, VIC, 3010, Australia.|Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC, 3052, Australia.
authors.forename: Haiyin
authors.initials: H
authors.lastname: Liu
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Rd, Parkville, VIC, 3010, Australia.
authors.forename: Claire
authors.initials: C
authors.lastname: Dumont
authors.affiliation: Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC, 3052, Australia. angus.johnston@monash.edu.|ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, 381 Royal Parade, Parkville, VIC, 3052, Australia. angus.johnston@monash.edu.
authors.forename: Angus P R
authors.initials: AP
authors.lastname: Johnston
authors.affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Rd, Parkville, VIC, 3010, Australia. j.mintern@unimelb.edu.au.
authors.forename: Justine D
authors.initials: JD
authors.lastname: Mintern
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology*; D005434:Flow Cytometry; D006801:Humans; D008856:Microscopy, Fluorescence; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000276:immunology*
chemical_list: D011956:Receptors, Cell Surface; D004247:DNA
keywords: Antibodies; Binding; Dendritic cell; Endocytosis; Flow cytometry; Fluorescence; Internalization; Receptor; Sensor