NADPH Oxidases pp 301-312 | Cite as

Kinetic Analysis of Phagosomal ROS Generation

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


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 



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.


  1. 1.
    Dupre-Crochet S, Erard M, Nusse O (2013) ROS production in phagocytes: why, when, and where? J Leukoc Biol 94:657–670CrossRefGoogle Scholar
  2. 2.
    Nauseef WM (2008) Biological roles for the NOX family NADPH oxidases. J Biol Chem 283:16961–16965CrossRefGoogle Scholar
  3. 3.
    O’Neill S, Brault J, Stasia MJ, Knaus UG (2015) Genetic disorders coupled to ROS deficiency. Redox Biol 6:135–156CrossRefGoogle Scholar
  4. 4.
    Imlay JA (2008) Cellular defenses against superoxide and hydrogen peroxide. Annu Rev Biochem 77:755–776CrossRefGoogle Scholar
  5. 5.
    Seider K, Heyken A, Lüttich A, Miramón P, Hube B (2010) Interaction of pathogenic yeasts with phagocytes: survival, persistence and escape. Curr Opin Microbiol 13:392–400CrossRefGoogle Scholar
  6. 6.
    Nault L, Bouchab L, Dupré-Crochet S, Nüsse O, Erard M (2016) Environmental effects on ROS-detection - learning from the phagosome. Antioxid Redox Signal 25:564–576CrossRefGoogle Scholar
  7. 7.
    Tlili A, Dupré-Crochet S, Erard M, Nüße O (2011) Kinetic analysis of phagosomal production of reactive oxygen species. Free Radic Biol Med 50:438–447CrossRefGoogle Scholar
  8. 8.
    Wardman P (2007) Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects. Free Radic Biol Med 43:995–1022CrossRefGoogle Scholar
  9. 9.
    Dewitt S, Laffafian I, Hallett MB (2003) Phagosomal oxidative activity during beta2 integrin (CR3)-mediated phagocytosis by neutrophils is triggered by a non-restricted Ca2+ signal: Ca2+ controls time not space. J Cell Sci 116:2857–2865CrossRefGoogle Scholar
  10. 10.
    Tlili A, Erard M, Faure MC, Baudin X, Piolot T, Dupre-Crochet S, Nusse O (2012) Stable accumulation of p67(phox) at the phagosomal membrane and ROS production within the phagosome. J Leukoc Biol 91:83–95CrossRefGoogle Scholar
  11. 11.
    Song ZM, Bouchab L, Hudik E, Le Bars R, Nüsse O, Dupré-Crochet S (2017) Phosphoinositol 3-phosphate acts as a timer for reactive oxygen species production in the phagosome. J Leukoc Biol 101:1155–1168CrossRefGoogle Scholar
  12. 12.
    Bernardo J, Long HJ, Simons ER (2010) Initial cytoplasmic and phagosomal consequences of human neutrophil exposure to Staphylococcus epidermidis. Cytom Part A 77A:243–252Google Scholar
  13. 13.
    Russell DG, VanderVen BC, Glennie S, Mwandumba H, Heyderman RS (2009) The macrophage marches on its phagosome: dynamic assays of phagosome function. Nat Rev Immunol 9:594–U84CrossRefGoogle Scholar
  14. 14.
    Vanderven BC, Yates RM, Russell DG (2009) Intraphagosomal measurement of the magnitude and duration of the oxidative burst. Traffic 10:372–378CrossRefGoogle Scholar
  15. 15.
    Kamen LA, Levinsohn J, Cadwallader A, Tridandapani S, Swanson JA (2008) SHIP-A increases early oxidative burst and regulates phagosome maturation in macrophages. J Immunol 180:7497–7505CrossRefGoogle Scholar
  16. 16.
    Chen X, Zhong Z, Xu Z, Chen L, Wang Y (2010) 2′,7′-Dichlorodihydrofluorescein as a fluorescent probe for reactive oxygen species measurement: forty years of application and controversy. Free Radic Res 44:587–604CrossRefGoogle Scholar
  17. 17.
    Kundu K, Knight SF, Willett N, Lee S, Taylor WR, Murthy N (2009) Hydrocyanines: a class of fluorescent sensors that can image reactive oxygen species in cell culture, tissue, and in vivo. Angew Chemie Int Ed Engl 48:299–303CrossRefGoogle Scholar
  18. 18.
    Erard M, Dupre-Crochet S, Nusse O (2018) Biosensors for spatiotemporal detection of reactive oxygen species in cells and tissues. Am J Physiol Integr Comp Physiol 314(5):R667–R683. CrossRefGoogle Scholar
  19. 19.
    Schwarzlander M, Dick TP, Meyer AJ, Morgan B (2016) Dissecting redox biology using fluorescent protein sensors. Antioxid Redox Signal 24:680–712CrossRefGoogle Scholar
  20. 20.
    Seider K, Brunke S, Schild L, Jablonowski N, Wilson D, Majer O, Barz D, Haas A, Kuchler K, Schaller M, Hube B (2011) The facultative intracellular pathogen Candida glabrata subverts macrophage cytokine production and phagolysosome maturation. J Immunol 187:3072–3086CrossRefGoogle Scholar

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

Personalised recommendations