Amplex red assay for measuring hydrogen peroxide production from caenorhabditis elegans
Open accessAbstract
Reagents such as Amplex ® Red have been developed for detecting hydrogen peroxide (H2O2) and are used to measure the release of H2O2 from biological samples such as mammalian leukocytes undergoing the oxidative burst.Caenorhabditis elegans is commonly used as a model host in the study of interactions with microbial pathogens and releases reactive oxygen species (ROS) as a component of its defense response.We adapted the Amplex ® Red Hydrogen Peroxide/Peroxidase Assay Kit to measure H2O2 output from live Caenorhabditis elegans exposed to microbial pathogens.The assay differs from other forms of ROS detection in the worm, like dihydrofluorescein dyes and genetically encoded probes such as HyPer, in that it generally detects released, extracellular ROS rather than intracellular ROS, though the distinction between the two is blurred by the fact that certain species of ROS, including H2O2, can cross membranes.The protocol involves feeding C. elegans on a lawn of the pathogen of interest for a period of time.The animals are then rinsed off the plates in buffer and washed to remove any microbes on their cuticle.Finally, the animals in buffer are distributed into 96-well plates and Amplex ® Red and horseradish peroxidase (HRP) are added.Any H2O2 released into the buffer by the worms will react with the Amplex ® Red reagent in a 1:1 ratio in the presence of HRP to produce the red fluorescent excitation product resorufin that can be measured fluorometrically or spectrophotometrically, and the amount of H2O2 released can be calculated by comparison to a standard curve.The assay is most appropriate for studies focused on released ROS, and its advantages include ease of use, the ability to use small numbers of animals in a plate reader assay in which measurements can be taken either fluorometrically or spectrophotometrically.
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Amplex Red is cell-impermeable and specifically detects H2O2; in the presence of HRP and H2O2, it is oxidized to red fluorescent 10-acetyl-3,7-dihydroxypenoxazine, making it optimal for extracellular H2O2.
“Amplex Red also is cell-impermeable but, in contrast to isoluminol, detects specifically H2O2 [362]. In the presence of HRP and H2O2, Amplex Red is oxidized to red fluorescent 10-acetyl-3,7-dihydroxypenoxazine [363]. In consequence, this probe is optimal for determination of specifically extracellular generation of H2O2.”
From Functions of ROS in Macrophages and Antimicrobial Immunity (2021) · cites this paper as [362]
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- Authors
- Karakuzu, O., Cruz, M., Liu, Y., & Garsin, D.
- Affiliations
- The University of Texas Health Science Center at Houston (US)
- The University of Texas MD Anderson Cancer Center (US)
- Corresponding author
- Garsin, Danielle
- Venue
- BIO-PROTOCOL
- Publisher
- Bio-Protocol, LLC
- Published
- 2019
- Volume
- 9
- Issue
- 21
- ISSN
- 2331-8325 (ISSN-L)
- PMC
- PMC7375442
- OpenAlex
- W2987943890
- Cited by
- 48
- References
- 20
- Citations by year
- 2026: 7 · 2025: 12 · 2024: 7 · 2023: 8 · 2022: 6 · 2021: 7 · 2020: 1
- FWCI
- 1.40
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- BIO-PROTOCOL (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Funders
- National Institutes of Health
- National Institute of Allergy and Infectious Diseases (R01AI076406)
- Topics
- Life Sciences › Immunology and Microbiology › Immunology › Invertebrate Immune Response Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
- Keywords
- hydrogen peroxide, Caenorhabditis elegans, reactive oxygen species, host-pathogen interaction, pathogenic microorganisms, respiratory burst, resorufin, horseradish peroxidase, fluorometric assay
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