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Neutrophil pp 301-324 | Cite as

Measurement of Respiratory Burst Products, Released or Retained, During Activation of Professional Phagocytes

  • Claes DahlgrenEmail author
  • Halla Björnsdottir
  • Martina Sundqvist
  • Karin Christenson
  • Johan Bylund
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2087)

Abstract

Activation of professional phagocytes, potent microbial killers of our innate immune system, is associated with an increased cellular consumption of molecular oxygen (O2). The O2 molecules consumed are reduced by electrons delivered by a membrane localized NADPH-oxidase that initially generate one- and two electron reduced superoxide anions (O2) and hydrogen peroxide (H2O2), respectively. These oxidants can then be processed into other highly reactive oxygen species (ROS) that can kill microbes, but that may also cause tissue destruction and drive other immune cells into apoptosis. The development of basic techniques to measure and quantify ROS generation by phagocytes is of great importance, and a large number of methods have been used for this purpose. A selection of methods (including chemiluminescence amplified by luminol or isoluminol, absorbance change following reduction of cytochrome c, and fluorescence increase upon oxidation of PHPA) are described in detail in this chapter with special emphasis on how to distinguish between ROS that are released extracellularly, and those that are retained within intracellular organelles. These techniques can be valuable tools in research spanning from basic phagocyte biology to diagnosis of diseases linked to the NADPH-oxidase and more clinically oriented research on innate immune mechanisms and inflammation.

Key words

Reactive oxygen species Superoxide anions Hydrogen peroxide Myeloperoxidase Intracellular NADPH-oxidase activity Plasma membrane NADPH-oxidase activity Subcellular granules Chemiluminescence Cytochrome c reduction PHPA oxidation 

Notes

Acknowledgments

This work was supported by the Swedish Research Council, the Swedish Society for Medical Research, the IngaBritt and Arne Lundberg Research Foundation, the Swedish state under the LUA-ALF and TUA agreements, the Swedish Heart- and Lung Foundation, and the King Gustaf V Memorial Foundation. We thank Maria Hjulström and Hülya Çevik-Aras for performing chemiluminescence determinations with the National diagnostic kit and L-012, respectively.

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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Claes Dahlgren
    • 1
    Email author
  • Halla Björnsdottir
    • 2
  • Martina Sundqvist
    • 1
  • Karin Christenson
    • 2
  • Johan Bylund
    • 2
  1. 1.Department of Rheumatology and Inflammation Research, Institute of MedicineSahlgrenska Academy at University of GothenburgGothenburgSweden
  2. 2.Department of Oral Microbiology and Immunology, Institute of OdontologySahlgrenska Academy at University of GothenburgGothenburgSweden

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