title: An improved method for differentiating cell-bound from internalized particles by imaging flow cytometry.
abstract: Recognition, binding, internalization, and elimination of pathogens and cell debris are important functions of professional as well as non-professional phagocytes. However, high-throughput methods for quantifying cell-associated particles and discriminating bound from internalized particles have been lacking. Here we describe a protocol for using imaging flow cytometry to quantify the attached and phagocytosed particles that are associated with a population of cells. Cells were exposed to fluorescent particles, fixed, and exposed to an antibody of a different fluorophore that recognizes the particles. The antibody is added without cell permeabilization, such that the antibody only binds extracellular particles. Cells with and without associated particles were identified by imaging flow cytometry. For each cell with associated particles, a spot count algorithm was employed to quantify the number of extracellular (double fluorescent) and intracellular (single fluorescent) particles per cell, from which the percent particle internalization was determined. The spot count algorithm was empirically validated by examining the fluorescence and phase contrast images acquired by the flow cytometer. We used this protocol to measure binding and internalization of the bacterium Neisseria gonorrhoeae by primary human neutrophils, using different bacterial variants and under different cellular conditions. The results acquired using imaging flow cytometry agreed with findings that were previously obtained using conventional immunofluorescence microscopy. This protocol provides a rapid, powerful method for measuring the association and internalization of any particle by any cell type.
authors.affiliation: Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, USA.
authors.forename: Asya
authors.initials: A
authors.lastname: Smirnov
authors.affiliation: Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Solga
authors.affiliation: Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, USA. Electronic address: jl7fj@virginia.edu.
authors.forename: Joanne
authors.initials: J
authors.lastname: Lannigan
authors.affiliation: Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, USA.
authors.forename: Alison K
authors.initials: AK
authors.lastname: Criss
mesh_terms: D001692:Biological Transport / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D009504:Neutrophils / Q000502:physiology; D010586:Phagocytes / Q000502:physiology; D010587:Phagocytosis / Q000502:physiology
chemical_list: D005456:Fluorescent Dyes
keywords: Attachment; Bacteria; Imaging flow cytometry; Internalization; Phagocytosis