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Quantitative Assessment of Neutrophil Phagocytosis Using Flow Cytometry

  • Pontus Nordenfelt
Part of the Methods in Molecular Biology book series (MIMB, volume 1124)

Abstract

Neutrophils have an incredible ability to find and eradicate intruders such as bacteria and fungi. They do this largely through the process of phagocytosis, where the target is internalized into a phagosome, and eventually destroyed by the hostile phagosomal environment. It is important to study phagocytosis in order to understand how neutrophils interact with various pathogens and how they respond to different stimuli. Here, I describe a method to study neutrophil phagocytosis of bacteria using flow cytometry. The bacteria are fluorescently labeled before being introduced to neutrophils. After phagocytosis, both any remaining extracellular bacteria and neutrophils are labeled using one-step staining before three-color analysis. To assess phagocytosis, first the average time it takes for the neutrophils to internalize all bound bacteria is determined. Experiments are then performed using that time point while varying the bacteria-to-neutrophil ratio for full control of the analysis. Due to the ease with which multiple samples can be analyzed, and the quantitative nature of flow cytometry, this approach is both reproducible and sensitive.

Keywords

Neutrophil Phagocytosis Flow cytometry Bacteria Opsonization Streptococcus pyogenes 

Notes

Acknowledgements

This work was supported by the Swedish Research Council (524-2011-891), Swedish Society of Medicine (SLS-173751), and the Blanceflor Foundation.

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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Pontus Nordenfelt
    • 1
  1. 1.Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical SchoolHarvard UniversityBostonUSA

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