Background

Interest in developing assays to study the amount and type of reactive oxygen species produced by C. elegans was primarily driven by the use of this model organism in studies focused on aging and infection. Oxidative damage caused by ROS has long been hypothesized to contribute to aging, and therefore measuring the intracellular oxidative environment and how it changes over time and in various aging mutants is relevant. Likewise, ROS are generated during the C. elegans immune response where they are capable of acting as signaling or effector molecules. Understanding the sources of ROS and being able to measure their magnitude is therefore of key importance (reviewed by [Labuschagne and Brenkman, 2013; McCallum and Garsin, 2016; Miranda-Vizuete and Veal, 2017]). Commonly used methods to measure ROS in C. elegans include dihydrofluorescein dyes, the genetically encoded HyPer probe, and the Amplex^® Red assay. Additional methods with more specific applications include MioSox, a modified dihydroethidium (DHE) that is specific for measuring ROS in the mitochondria, and the Grx1-roGFP2 genetically-encoded probe that measures changes in redox potential (Labuschagne and Brenkman, 2013). All current methodologies that measure ROS have caveats that must be carefully considered when deciding what method to use and how to interpret the results.

The dihydrofluorescein dyes, including 2,7-dichlorodihydrofluorescein diacetate (H₂DCFDA) commonly used in C. elegans, are colorless and membrane permeable in the reduced form, but become fluorescent and non-membrane permeable when oxidized. Therefore, these dyes can penetrate the cytoplasmic membrane and provide a decent measure of intracellular ROS (Labuschagne and Brenkman, 2013). However, to measure the fluorescence from C. elegans using a plate reader, a large number of animals (1,000) are required (Schulz et al., 2007; Ewald et al., 2017). Alternatively, animals can be visualized using fluorescence microscopy (Sem and Rhen, 2012). Two disadvantages to H₂DCFDA include the fact that it is not specific to H₂O₂, and it may exert free radical properties, contributing to the generation of ROS in addition to its measurement (Labuschagne and Brenkman, 2013; Dikalov and Harrison, 2014).

HyPer, in contrast, is considered very specific to H₂O₂. It consists of a cpYFP (circularly permuted yellow fluorescent protein) inserted into the regulatory domain of OxyR-RD and placed under the control of aC. elegans promoter. Both tissue-specific promoters and non-specific promoters have been utilized in the worm depending on the application (Back et al., 2012; Knoefler et al., 2012). The sensor works based on the formation of an intramolecular disulfide bond that occurs upon interaction with H₂O₂. The strength of the two fluorescent emission peaks released by HyPer changes upon formation of the disulfide bond; one decreases and the other increases in a proportionate manner (Belousov et al., 2006). The advantage of such a ratiometric approach is that quantification can be done independently of the expression levels of the protein and therefore no normalization is required (Labuschagne and Brenkman, 2013). An additional advantage is the ability to monitor hydrogen peroxide levels over time and observe both increases and decreases. Disadvantages include the fact that HyPer is sensitive to pH changes and overexpression of these probes can significantly affect H₂O₂ redox signaling of the cell potentially confounding results. Furthermore, measuring the emission peaks requires sophisticated microscopy of individual animals or large numbers of animals (1,000/sample) to obtain accurate results from a plate reader (Labuschagne and Brenkman, 2013; Dikalov and Harrison, 2014; Ewald et al., 2017).

The Amplex^® Red assay is based on the oxidation of 10-acetyl-3,7- dihydroxypenoxazine, which is catalyzed by horseradish peroxidase (HRP) in the presence of H₂O₂ to produce a red fluorescent oxidation product, resorufin, at a 1:1 ratio (Figure 1). Resorufin can be measured both fluorometrically and spectrophotometrically, though the fluorescent measurements are more sensitive and can detect lower levels of H₂O₂ (Mohanty et al., 1997). The Amplex^® Red Hydrogen Peroxide/Peroxidase Assay Kit was first adapted to C. elegans by our group to measure the output of ROS following exposure to microbial pathogens (Chavez et al., 2007; Chavez et al., 2009; Tiller and Garsin, 2014; van der Hoeven, et al., 2015; Liu et al., 2019). The assay measures the amount of H₂O₂ excreted by the worm into the surrounding medium. The major source of ROS during this response appears to be an NADPH oxidase called BLI-3, which is found in the hypodermis (skin), intestine and pharynx (Edens et al., 2001; Chavezet al., 2007 and 2019; van der Hoeven et al., 2015). BLI-3 is a member of the dual oxidase (DUOX) family of proteins, which all feature a peroxidase domain in addition to an NADPH oxidase domain that generates H₂O₂ rather than superoxide (Aguirre and Lambeth, 2010). Bioinformatic predictions and experimental evidence indicate that BLI-3 is located in the cytoplasmic membranes of the tissue in which it is produced and oriented in a manner such that H₂O₂ will be excreted into extracellular environments (Edens et al., 2001; Chavez et al., 2009; Aguirre and Lambeth, 2010; Moribe et al., 2012; van der Hoeven et al., 2015). Based on the localization of BLI-3, H₂O₂ is released from the hypodermal surface and into the lumen of the GI tract whereupon it is excreted due to the defecation cycle that the animals continuously undergo. When measuring H₂O₂ from animals exposed to pathogen, the levels can be significantly, but not completely, suppressed by the addition of an NADPH oxidase inhibitor (Chavez et al., 2007 and 2009). These results suggest that there are additional sources of ROS during infection that are contributing the pool of H₂O₂ that is being measured. A likely source is the mitochondria, which are known to become dysfunctional during pathogen assault (Pellegrino et al., 2014), and ROS generated from this source in the form of H₂O₂ would be able to cross membranes and contribute to the extracellular pools. Advantages to using the Amplex^® Red assay for measuring ROS in the worm include the fact that it is specific to H₂O₂, it can be done on samples containing as few as 30 worms in a plate reader, and it does not require sophisticated genetics or microscopy (Labuschagne and Brenkman, 2013; Tiller and Garsin, 2014). Disadvantages include the fact that there is no tissue specificity and one must be careful to maintain samples in the dark, as resorurfin is light sensitive (Labuschagne and Brenkman, 2013). Herein, we present a detailed protocol of our adaption of the Amplex^® Red Hydrogen Peroxide/Peroxidase Assay Kit to measuring H₂O₂ output from the worm following pathogen exposure.