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Quantification of Chemotaxis or Respiratory Burst Using Ex Vivo Culture-Derived Murine Neutrophils

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Neutrophil

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2087))

Abstract

Two critical functional responses of neutrophils are chemotaxis, a response driven by concentration gradients of chemokines released by infected or inflamed tissues, and production of reactive oxygen species (ROS), molecules essential to their capacity to kill pathogens. Assays to accurately test each response have been important to assess efficacies of pharmaceuticals predicted to block recruitment of neutrophils or attenuate their ROS production. Identified antagonists to neutrophil functions may help to reduce tissue damage following inflammation. Described are detailed assays to test these functions, along with steps to generate neutrophils from ex vivo-cultured murine bone marrow that produce robust responses in either assay. The first function protocol details a quantitative assay for chemotaxis that involves culture plates with dual chamber wells that separate cells from a chemokine with small pore-sized membranes. Quantitative measurements of cell numbers in the chemokine-containing chamber are performed with either fluorescence or luminescence detection reagents, which provide signals directly proportional to the numbers of migrated cells. Multiwell plates are used for rapidly testing a variety of conditions and/or chemoattractants. Described in the second function protocol is an assay to measure ROS produced by stimulated neutrophils, again using a multiwell platform for rapid, quantitative measurements of several conditions simultaneously.

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Acknowledgments

This work was supported in part by the National Heart, Lung, and Blood Institute at the National Institutes of Health (Academic Research Enhancement Award grant No. 1R15HL104593 to P.G.).

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Authors and Affiliations

  1. Department of Biological Sciences, University of Massachusetts Lowell, Lowell, MA, USA

    Klaudia Szymczak, Margery G. H. Pelletier & Peter C. W. Gaines

Authors
  1. Klaudia Szymczak
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  2. Margery G. H. Pelletier
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  3. Peter C. W. Gaines
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Corresponding author

Correspondence to Peter C. W. Gaines .

Editor information

Editors and Affiliations

  1. Department of Microbiology and Immunology, Montana State University, Bozeman, MT, USA

    Mark T. Quinn

  2. Laboratory of Bacteriology, Rocky Mountain Laboratories Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA

    Frank R. DeLeo

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Szymczak, K., Pelletier, M.G.H., Gaines, P.C.W. (2020). Quantification of Chemotaxis or Respiratory Burst Using Ex Vivo Culture-Derived Murine Neutrophils. In: Quinn, M., DeLeo, F. (eds) Neutrophil. Methods in Molecular Biology, vol 2087. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0154-9_7

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  • DOI: https://doi.org/10.1007/978-1-0716-0154-9_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0153-2

  • Online ISBN: 978-1-0716-0154-9

  • eBook Packages: Springer Protocols

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