Scientific article
Open access

Detecting, visualizing and quantitating the generation of reactive oxygen species in an amoeba model system

Published inJournal of visualized experiments, no. 81, e50717
Publication date2013

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

  • Dictyostelium/chemistry/metabolism
  • Ethidium/analogs & derivatives/chemistry
  • Fluoresceins/chemistry
  • Fluorescent Dyes/chemistry
  • Hydrogen Peroxide/analysis/metabolism
  • Microscopy, Fluorescence/methods
  • Oxazines/chemistry
  • Phagosomes/chemistry/metabolism
  • Reactive Oxygen Species/analysis/metabolism
Citation (ISO format)
ZHANG, Xuezhi, SOLDATI, Thierry. Detecting, visualizing and quantitating the generation of reactive oxygen species in an amoeba model system. In: Journal of visualized experiments, 2013, n° 81, p. e50717. doi: 10.3791/50717
Main files (1)
Article (Published version)
ISSN of the journal1940-087X

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