Using Imaging Flow Cytometry to Quantify Neutrophil Phagocytosis

Asya Smirnov, Michael D. Solga, Joanne Lannigan, Alison K. Criss

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

3 Scopus citations

Abstract

Neutrophils are professional phagocytes that are important for innate host defenses against pathogens and resolution of inflammation. Traditionally, the phagocytic capacity of neutrophils was quantified by enumeration of cells containing either internalized or bound bacteria or other cargo from a series of microscopic images. Here we describe an imaging flow cytometry-based protocol and analysis method for quantifying the binding and uptake of Neisseria gonorrhoeae by primary adherent human neutrophils. Imaging flow cytometry combines the capacity for quantitative, high-throughput analysis of tens of thousands of cells per condition, with the imaging power of fluorescence microscopy. Here, all bacteria are labeled with Tag-it Violet™ and bound bacteria are differentially stained with a DyLight™ 650-conjugated antibody. Images are analyzed using spot count and other algorithms. Outputs include the percent of neutrophils associated with bacteria, the percent of neutrophils with internalized bacteria, and the percent of internalized bacteria. This basic protocol can be adapted to a variety of particle types and can be used for multiplex analysis in combination with staining for different neutrophil surface and intracellular markers.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages127-140
Number of pages14
DOIs
StatePublished - 2020

Publication series

NameMethods in Molecular Biology
Volume2087
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Bacteria
  • Binding
  • Fluorescence
  • Imaging flow cytometry
  • Internalization
  • Neutrophils
  • Phagocytosis

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