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NADPH Oxidases pp 301-312 | Cite as

Kinetic Analysis of Phagosomal ROS Generation

  • Sophie Dupré-Crochet
  • Marie Erard
  • Oliver NüβeEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1982)

Abstract

Phagosomal ROS generation is critical for our immune defense against microbial infections. Quantitative assessment of phagosomal ROS production is required to understand the complex relationship between the phagocyte and the microbe, in particular for pathogens that resist phagosomal destruction. ROS detection is difficult due to the transient nature of the reactive species and their multiple interactions with the environment. Direct labeling of phagocytic prey with a ROS-sensitive dye allows to target the dye into the phagosome and to follow the kinetics of phagosomal ROS production on a single phagosome base. Here we describe the basic labeling procedure, the quality assessment, and the imaging technique to achieve this kinetic analysis.

Key words

NADPH oxidase Phagosome Microbial killing Yeast ROS-sensitive dyes Covalent labeling DCF DCFH2-SE Imaging Flow cytometry 

Notes

Acknowledgments

This work was supported by grants from the FRM (Foundation for Medical Research, DCM20121225747). We wish to thank our past colleagues who have contributed to this method and Elodie Hudik for her excellent technical assistance.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Sophie Dupré-Crochet
    • 1
  • Marie Erard
    • 1
  • Oliver Nüβe
    • 1
    Email author
  1. 1.LCP, CNRS UMR 8000, Université Paris-Sud, Université Paris-SaclayOrsayFrance

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