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

  • Johan Bylund
  • Halla Björnsdottir
  • Martina Sundqvist
  • Anna Karlsson
  • Claes Dahlgren
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1124)

Abstract

Activation of professional phagocytes, potent microbial killers of our innate immune system, is associated with an increase in cellular consumption of molecular oxygen (O2). The consumed O2 is utilized by an NADPH-oxidase to generate highly reactive oxygen species (ROS) by a one electron reduction, initially generating superoxide anion (O2 ) that then dismutates to hydrogen peroxide (H2O2). The ROS are strongly bactericidal molecules but may also cause tissue destruction, and are capable of driving immune competent cells of both the innate and the adaptive immune systems into apoptosis. The development of basic techniques to measure/quantify ROS generation by phagocytes during activation of the respiratory burst 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, the absorbance change following reduction of cytochrome c, and the 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 more clinically oriented research on innate immune mechanisms and inflammation.

Keywords

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

Notes

Acknowledgements

This work was supported by the Swedish Medical Research Council, the Swedish Society for Medical Research, the IngaBritt and Arne Lundberg Research Foundation, the Swedish state under the LUA-ALF agreement, 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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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Johan Bylund
    • 1
  • Halla Björnsdottir
    • 1
  • Martina Sundqvist
    • 1
  • Anna Karlsson
    • 1
  • Claes Dahlgren
    • 1
  1. 1.The Phagocyte Research Group, Department of Rheumatology and Inflammation ResearchUniversity of GothenburgGothenburgSweden

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