Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Abstract

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, mediumthroughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H 2 O 2 production, respectively. Video LinkThe video component of this article can be found at

The small heat shock protein Hsp27 binds α-synuclein fibrils, preventing elongation and cytotoxicity

“…The dye dihydroethidium (DHE) has been used extensively to monitor the intracellular production of reactive oxygen species (ROS), specifically superoxide (52)(53)(54), as a measure of cellular toxicity. This method was therefore chosen to measure the toxicity of α-syn fibrils when added exogenously to cells.…”

“…Quantitative co-localization and wavelength analyses were completed using custom scripts written for Python 2.7, and the localisations were binned into low, medium and high wavelengths using GraphPad Prism v 5 (GraphPad Software Inc., San Diego, USA).Dihydroethidium (DHE) assay to monitor cellular ROS production A DHE cell-based assay, similar to that described previously (52,71), was used to investigate the toxicity associated α-syn aggregates when added exogenously to cells. Neuro-2a (N2a) o C. The cells were seeded into 8-well chamber μSlides (Ibidi, Martinsried, Germany) and cultured to 60% confluency.…”

“…Images were taken every 30 s for 15 min using an epifluorescence microscope (Leica DMi8 fluorescence microscope; Wetzlar, Germany) to quantify the amount of DHE (excitation 325 -375 nm and emission 435 -485 nm) and its oxidised product (i.e. ethidium, excitation 540 -580 nm and emission 592 -668 nm) as a measure of ROS production in cells (52). The solutions were then added to cells, and images were taken every 30 s for a further 15 min.…”

The Impact of Protozoan Predation on the Pathogenicity of Vibrio cholerae

“…ROS/RNS are oxidative species that can directly damage the DNA of microorganisms, thereby acting as a natural antimicrobial barrier. Human professional phagocytes and amoeba are known to produce ROS/RNS such as nitric oxide (NO) and hydrogen peroxide (H 2 O 2 ) inside phagosomes as an antibacterial strategy (Zhang and Soldati, 2013;Di Meo et al, 2016). Indeed, the production of ROS in the phagosome of the amoeba Dictyostelium discoideum has been visualized and quantified (Zhang and Soldati, 2013).…”

“…Human professional phagocytes and amoeba are known to produce ROS/RNS such as nitric oxide (NO) and hydrogen peroxide (H 2 O 2 ) inside phagosomes as an antibacterial strategy (Zhang and Soldati, 2013;Di Meo et al, 2016). Indeed, the production of ROS in the phagosome of the amoeba Dictyostelium discoideum has been visualized and quantified (Zhang and Soldati, 2013). Furthermore, it is known that the intestinal epithelial layer also produces NO by the induction of oxide synthases (iNOS) (Eckmann et al, 2000).…”