title: Monitoring virus entry into living cells using DiD-labeled dengue virus particles.
abstract: A variety of approaches can be applied to investigate the multiple steps and interactions that occur during virus entry into the host cell. Single-virus tracking is a powerful real-time imaging technique that offers the possibility to monitor virus-cell binding, internalization, intracellular trafficking behavior, and the moment of membrane fusion of single virus particles in living cells. Here we describe the development and applications of a single-virus tracking assay based on the use of DiD-labeled dengue virus (DENV) in BS-C-1 cells. In addition - and using the same experimental setup - we present a binding and fusion assay that can be used to obtain a rapid insight into the relative extent of virus binding to the cell surface and membrane fusion. Details of virus labeling and characterization, microscopy setup, protocols, data analysis, and hints for troubleshooting are described throughout the paper.
authors.affiliation: Department of Medical Microbiology, Molecular Virology Section, University Medical Center Groningen, University of Groningen, 9700 RB Groningen, The Netherlands.
authors.forename: Nilda V
authors.initials: NV
authors.lastname: Ayala-Nuñez
authors.forename: Jan
authors.initials: J
authors.lastname: Wilschut
authors.forename: Jolanda M
authors.initials: JM
authors.lastname: Smit
mesh_terms: D000818:Animals; D002232:Carbocyanines / Q000737:chemistry*; D002478:Cells, Cultured; D003716:Dengue Virus / Q000502:physiology*; D004705:Endocytosis; D005456:Fluorescent Dyes / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D018715:Microscopy, Video / Q000379:methods; D013194:Staining and Labeling / Q000379:methods; D014771:Virion / Q000737:chemistry* / Q000302:isolation & purification; D053586:Virus Internalization
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; C024286:3,3'-dioctadecylindocarbocyanine