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Quantification of Phagocytosis Using Flow Cytometry

de Neergaard, Therese LU and Nordenfelt, Pontus LU orcid (2023) In Methods in molecular biology (Clifton, N.J.) p.221-234
Abstract

Phagocytosis is relevant for many research fields and is often measured as a functional outcome. However, accurate quantification can be challenging, and many researchers find it difficult to study in a robust manner. There are many ways to measure phagocytosis, but what is often overlooked is the importance of experimental design and how the analysis is planned and performed. Experimental factors like reaction volume, time, and phagocyte-prey concentrations often have a large impact on the outcome, as is the choice of detection strategy with different fluorescent or colorimetric labels of prey and phagocyte. By using dose-response curve principles for both experimental design and analysis, it is possible to increase the sensitivity and... (More)

Phagocytosis is relevant for many research fields and is often measured as a functional outcome. However, accurate quantification can be challenging, and many researchers find it difficult to study in a robust manner. There are many ways to measure phagocytosis, but what is often overlooked is the importance of experimental design and how the analysis is planned and performed. Experimental factors like reaction volume, time, and phagocyte-prey concentrations often have a large impact on the outcome, as is the choice of detection strategy with different fluorescent or colorimetric labels of prey and phagocyte. By using dose-response curve principles for both experimental design and analysis, it is possible to increase the sensitivity and robustness, leading to accurate quantification of phagocytosis that is comparable across experiments and systems.Here, we describe how to quantify phagocytosis using flow cytometry with a robust, high-throughput, and easy-to-use approach. The prey is first fluorescently double stained, followed by optional opsonization before being introduced to the phagocyte in a wide range of ratios. After incubation, data is acquired through flow cytometry. It can be assessed on both the population and single-cell level of the phagocytes, separating adhesion and internalization. As an example, we provide an experimental protocol for studying phagocytosis of opsonized Streptococcus pyogenes using the THP-1 cell line. This approach is easily incorporated into most existing phagocytosis assays and allows for reproducible results with high sensitivity.

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Please use this url to cite or link to this publication:
author
and
organization
publishing date
type
Chapter in Book/Report/Conference proceeding
publication status
published
subject
keywords
Flow Cytometry/methods, Phagocytosis, Phagocytes/physiology, Coloring Agents, Streptococcus pyogenes
host publication
Bacterial Pathogenesis : Methods and Protocols - Methods and Protocols
series title
Methods in molecular biology (Clifton, N.J.)
editor
Nordenfelt, Pontus and Collin, Mattias
edition
2
pages
14 pages
external identifiers
  • scopus:85160771390
  • pmid:37258971
ISSN
1940-6029
ISBN
978-1-0716-3242-0
DOI
10.1007/978-1-0716-3243-7_15
language
English
LU publication?
yes
additional info
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
id
1e40ed28-7a7d-44f4-a54d-74e04eb2b10c
date added to LUP
2023-06-09 14:15:15
date last changed
2025-05-04 09:31:25
@inbook{1e40ed28-7a7d-44f4-a54d-74e04eb2b10c,
  abstract     = {{<p>Phagocytosis is relevant for many research fields and is often measured as a functional outcome. However, accurate quantification can be challenging, and many researchers find it difficult to study in a robust manner. There are many ways to measure phagocytosis, but what is often overlooked is the importance of experimental design and how the analysis is planned and performed. Experimental factors like reaction volume, time, and phagocyte-prey concentrations often have a large impact on the outcome, as is the choice of detection strategy with different fluorescent or colorimetric labels of prey and phagocyte. By using dose-response curve principles for both experimental design and analysis, it is possible to increase the sensitivity and robustness, leading to accurate quantification of phagocytosis that is comparable across experiments and systems.Here, we describe how to quantify phagocytosis using flow cytometry with a robust, high-throughput, and easy-to-use approach. The prey is first fluorescently double stained, followed by optional opsonization before being introduced to the phagocyte in a wide range of ratios. After incubation, data is acquired through flow cytometry. It can be assessed on both the population and single-cell level of the phagocytes, separating adhesion and internalization. As an example, we provide an experimental protocol for studying phagocytosis of opsonized Streptococcus pyogenes using the THP-1 cell line. This approach is easily incorporated into most existing phagocytosis assays and allows for reproducible results with high sensitivity.</p>}},
  author       = {{de Neergaard, Therese and Nordenfelt, Pontus}},
  booktitle    = {{Bacterial Pathogenesis : Methods and Protocols}},
  editor       = {{Nordenfelt, Pontus and Collin, Mattias}},
  isbn         = {{978-1-0716-3242-0}},
  issn         = {{1940-6029}},
  keywords     = {{Flow Cytometry/methods; Phagocytosis; Phagocytes/physiology; Coloring Agents; Streptococcus pyogenes}},
  language     = {{eng}},
  pages        = {{221--234}},
  series       = {{Methods in molecular biology (Clifton, N.J.)}},
  title        = {{Quantification of Phagocytosis Using Flow Cytometry}},
  url          = {{http://dx.doi.org/10.1007/978-1-0716-3243-7_15}},
  doi          = {{10.1007/978-1-0716-3243-7_15}},
  year         = {{2023}},
}