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

Summary

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

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, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H₂O₂ production, respectively.

Introduction

Reactive oxygen species (ROS) are involved in a wide variety of biological processes such as host defense, signaling, tissue development and response to injury as well as hypertension and cancer. The well-studied phagosomal NADPH oxidase machinery is dedicated to rapid ROS generation, known as the oxidative burst, to kill bacteria ingested in neutrophils' phagosomes¹. In addition, leakage of electrons as a byproduct of the mitochondrial respiratory chain was previously thought to be responsible only for an unregulated source of ROS. But recently, it was identified as an important mechanism to contribute to intraphagosomal killing of bacteria in mouse ma....

Protocol

1. Visualization and Qualitative Measurement of ROS Production in Phagosomes

The OBG-coated beads should be prepared in advance. The beads coating procedures and agar overlay technique are adapted from published references^5,14.

1. Add 1 ml suspension of 3.0 μm carboxylated silica beads (about 1.8 x 10⁹ beads) into an 1.5 ml tube, wash the beads 3x with 1 ml of PBS by quick spin and vortexing.
2. Resuspend the beads in 1 ml of PBS containing 25 mg/ml cyanamide (to activate the carboxylated silica beads to covalently bind prelabeled BSA), incubate on a wheel for 15 min.
3. Wash 3x with 1 ml of....

Results

The generation of ROS in phagosomes can be visualized qualitatively and dynamically by microscopy (File S1). The red fluorescence emitted by Alexa fluor 594 is pH-insensitive and remains constant in the phagosomal environment, while oxidation of OBG increases its fluorescence in the green channel. The emission spectra of in vitro oxidized and nonoxidized OBG-coated beads are compared in Figure 1B, showing a significant increase in intensity after oxidation. To facilitate visuali.......

Discussion

Compared to the previously described methods, the OBG assay dynamically visualizes the process of phagosomal ROS generation at the single cell level, instead of measuring an average ROS signal from a population of cells. Such population-averaging methods tend to obscure critical information caused by nonsynchronous phagocytosis. We successfully adapted DHE and AUR assays to Dictyostelium and optimized the protocols. Most importantly, we specified the types and subcellular localizations of ROS measured by these t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
REAGENTS
OxyBURST Green H2HFF BSA	Invitrogen	O-13291
Alexa Fluor 594	Invitrogen	A-20004
Carboxylated silica beads	Kisker Biotech	PSi-3.0COOH
HL5C medium	ForMedium	HLC0102
LoFlo medium	ForMedium	LF1001
Bacto agar	BD	204010
Dihydroethidium	Sigma	37291-25MG
Amplex UltraRed	Invitrogen	A36006
Horseradish peroxidase	Roche	10108090001
Diethyldithiocarbamate (DEDTC)	Sigma	D3506-100G
Catalase	Sigma	C9322-1G
Cyanamide	Sigma	187364-25G
Lipopolysaccharides	Sigma	L2630-25G
EQUIPMENT
ibidi μ-Dish 35 mm, high	ibidi	81156	Bottom made of optically clear plastic
MatTek dish 35 mm	MatTek corporation	P35G-1.5-14-C	Bottom made of a glass coverslip
Scepter Cell Counter	Merck Millipore	PHCC00000
White 96 well plate	Nunc	236108
Centrifuge	SORVALL	Legend RT
Microplate reader	BioTek	Synergy Mx