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Here we present a protocol for high\u2010throughput analysis of particle uptake by imaging flow cytometry, using the bacterium <jats:italic>Neisseria gonorrhoeae<\/jats:italic> attached to and internalized by neutrophils as an example. Cells are exposed to fluorescently labeled bacteria, fixed, and stained with a bacteria\u2010specific antibody of a different fluorophore. Thus, in the absence of a permeabilizing agent, extracellular bacteria are double\u2010labeled with two fluorophores while intracellular bacteria remain single\u2010labeled. A spot count algorithm is used to determine the number of single\u2010 and double\u2010labeled bacteria in individual cells, to calculate the percent of cells associated with bacteria, percent of cells with internalized bacteria, and percent of cell\u2010associated bacteria that are internalized. These analyses quantify bacterial association and internalization across thousands of cells and can be applied to diverse experimental systems. \u00a9 2017 by John Wiley &amp; Sons, Inc.<\/jats:p>","DOI":"10.1002\/cpcy.19","type":"journal-article","created":{"date-parts":[[2017,4,5]],"date-time":"2017-04-05T14:10:11Z","timestamp":1491401411000},"source":"Crossref","is-referenced-by-count":14,"title":["High\u2010Throughput Particle Uptake Analysis by Imaging Flow Cytometry"],"prefix":"10.1002","volume":"80","author":[{"given":"Asya","family":"Smirnov","sequence":"first","affiliation":[{"name":"Department of Microbiology, Immunology, and Cancer Biology, University of Virginia  Charlottesville Virginia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael D.","family":"Solga","sequence":"additional","affiliation":[{"name":"Office of Research Core Administration University of Virginia  Charlottesville Virginia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joanne","family":"Lannigan","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology, and Cancer Biology, University of Virginia  Charlottesville Virginia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alison K.","family":"Criss","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Immunology, and Cancer Biology, University of Virginia  Charlottesville 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