We are grateful to Drs Karl-Heinz Krause and Vincent Jaquet for help and advice to 
set up these protocols, and also thank Christoph Bauer and J&eacute;r&ocirc;me Bosset from the Bioimaging Platform of the NCCR and Dr. Navin Gopaldass for their technical support. The research is supported by a ProDoc grant of the Swiss National Science Foundation.

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 references5,14.
<ol>
	<li>Add 1 ml suspension of 3.0 &#956;m 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.</li>
	<li>Resuspend the beads in 1 ml of PBS containing 25 mg/ml c...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agar-overlay+immunofluorescence:+high-resolution+studies+of+cytoskeletal+components+and+their+changes+during+chemotaxis.">Agar-overlay immunofluorescence: high-resolution studies of cytoskeletal components and their changes during chemotaxis.</a> Methods Cell Biol. 28, 347-356 (1987).</li><li>Fey, P., Kowal, A. S., Gaudet, P., Pilcher, K. E., Chisholm, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+growth+and+development+of+Dictyostelium+discoideum.">Protocols for growth and development of Dictyostelium discoideum.</a> Nat. Protoc. 2, 1307-1316 (2007).</li><li>Bloomfield, G., Pears, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+signalling+required+for+multicellular+development+of+Dictyostelium.">Superoxide signalling required for multicellular development of Dictyostelium.</a> J. Cell. Sci. 116, 3387-3397 (2003).</li><li>Dieckmann, R., Gopaldass, N., Escalera, C., Soldati, T., Deretic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagosomes+and+Phagosomes.">Autophagosomes and Phagosomes.</a> Methods in Molecular Biology. 445, 317-328 (2008).</li><li>Amir, Y., Edward, O. -. 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No conflicts of interest declared.

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...

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&#39; 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 macrophages2. In recent years, the social amoeba, Dictyostelium, has become a powerful and popular model to study cell intrinsic mechanisms of the innate immune response. Indeed, Dictyostelium and human phagocytes share a surprisingly high level of conservation in molecular machineries responsible for bacteria sensing, engulfment and killing3,4. The homologs of proteins and enzymes related to ROS production or detoxification, such as NADPH oxidases, catalases, superoxide dismutases, can be found in both human and Dictyostelium. As the best studied amoeba, Dictyostelium presents several unique advantages over mammalian model system. They grow at room temperature without the need for CO2, with a doubling time of 8-10 hr. They can be easily kept as adherent or suspension cultures. In addition, thanks to their fully sequenced and annotated haploid genome, and to easy genetic manipulation, Dictyostelium has become a very attractive experimental model organism.
In previous studies, various chlorinated and fluorinated derivatives of fluorescein (collectively known as OxyBurst Green, OBG) that emit fluorescence after oxidation by ROS, have been used as a ROS reporter. Cell-permeant esterified derivatives are used to measure cytoplasmic ROS, whereas cell-impermeant dextran- or protein-coupled derivatives are used to measure extracellular ROS. In particular, OBG BSA-coated beads have already been used to detect phagosomal ROS production in mammalian cells5. However, the microplate reader-based approach can only give an averaged ROS generation curve from a population of cells. With the present protocol, by using Dictyostelium , a professional phagocyte, and optimized experimental conditions, we obtain stable and efficient phagocytosis without conjugating any opsonin onto the beads. DHE has been used in various model systems, such as mammalian neutrophils and macrophages, to detect ROS production6-9. Meanwhile, there are some controversies over the specificity and sensitivity of the method8,10. As an improved version of Amplex Red, the fluorescence intensity of AUR is less sensitive to pH, which makes it more suitable to measure ROS in nonneutral or weakly acidic milieus. AUR has been recently applied in several mammalian systems11,12, but its use in nonmammalian models, which quite often require weakly acidic growth media, has not been reported yet. In addition, there is no published protocol to quantitatively and dynamically measure ROS production and localization in the social amoeba Dictyostelium.
The goal of the presented set of protocols is to provide an easy and versatile solution to monitor various ROS and their localization, and further give insights into ROS-related cellular mechanisms. For the OBG assay, we used live microscopy to monitor the whole process of phagosomal ROS generation after uptake of OBG-coated beads by Dictyostelium cells, and provided a new approach to study the mechanism of intraphagosomal killing of bacteria. We have optimized medium-throughput DHE and AUR assays in Dictyostelium, to measure intracellular superoxide and extracellular H2O2 production, respectively, by using LPS as a potent ROS stimulator. Note that LPS was recently shown to increase the bactericidal activity of Dictyostelium towards phagocytosed bacteria13. In addition, treatment with DEDTC and catalase explicitly confirmed that these two methods specifically measure different types and subcellular localizations of ROS in Dictyostelium. Finally, the OBG assay can be adapted to any adherent phagocytic cell, by further opsonizing the beads with ligands for phagocytic receptors of animal cells. In principle, the DHE and AUR assays can also be fine-tuned for measurements in any adherent or nonadherent cell under appropriate experimental conditions.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>OxyBURST Green H2HFF BSA</td>
			<td>Invitrogen</td>
			<td>O-13291</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>A-20004</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxylated silica beads</td>
			<td>Kisker Biotech</td>
			<td>PSi-3.0COOH</td>
			<td></td>
		</tr>
		<tr>
			<td>HL5C medium</td>
			<td>ForMedium</td>
			<td>HLC0102</td>
			<td></td>
		</tr>
		<tr>
			<td>LoFlo medium</td>
			<td>ForMedium</td>
			<td>LF1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>BD</td>
			<td>204010</td>
			<td></td>
		</tr>
		<tr>
			<td>Dihydroethidium</td>
			<td>Sigma</td>
			<td>37291-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex UltraRed</td>
			<td>Invitrogen</td>
			<td>A36006</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase</td>
			<td>Roche</td>
			<td>10108090001</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyldithiocarbamate (DEDTC)</td>
			<td>Sigma</td>
			<td>D3506-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma</td>
			<td>C9322-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanamide</td>
			<td>Sigma</td>
			<td>187364-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides</td>
			<td>Sigma</td>
			<td>L2630-25G</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>ibidi &#956;-Dish 35 mm, high</td>
			<td>ibidi</td>
			<td>81156</td>
			<td>Bottom made of optically clear plastic</td>
		</tr>
		<tr>
			<td>MatTek dish 35 mm</td>
			<td>MatTek corporation</td>
			<td>P35G-1.5-14-C</td>
			<td>Bottom made of a glass coverslip</td>
		</tr>
		<tr>
			<td>Scepter Cell Counter</td>
			<td>Merck Millipore</td>
			<td>PHCC00000</td>
			<td></td>
		</tr>
		<tr>
			<td>White 96 well plate</td>
			<td>Nunc</td>
			<td>236108</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>SORVALL</td>
			<td>Legend RT</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>BioTek</td>
			<td>Synergy Mx</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

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.

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...

Watch this Scientific Journal Video about Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System at JoVE.com

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

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.

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

detecting-visualizing-quantitating-generation-reactive-oxygen-species

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JoVE Journal

Biology

16.0K visualizações.  University of Geneva.  Nós adaptamos um conjunto de protocolos para a medição de espécies reativas de oxigênio (ROS) que podem ser aplicados em vários modelos celulares ameba e mamíferos para estudos qualitativos e quantitativos. 