Abstract
Reactive oxygen species (ROS) are potent signaling molecules with critical roles in cellular pathology and homeostasis. They are produced in all cell types via a diverse array of cellular machinery, giving rise to an equally diverse repertoire of molecular effects. These range from cytotoxic killing of microbes to alteration of the cellular transcriptional response to stress. Despite their importance, research into ROS has been difficult given their inherent instability and transient signaling properties. Herein we describe methods for the use of the redox-sensitive probe hydro-Cy3 for the detection and quantification of ROS both in vitro and in vivo.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Yang Y, Bazhin AV, Werner J et al (2013) Reactive oxygen species in the immune system. Int Rev Immunol 32:249–270
Bogdan C, Röllinghoff M, Diefenbach A (2000) Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. Curr Opin Immunol 12:64–76
Dahlgren C, Karlsson A (1999) Respiratory burst in human neutrophils. J Immunol Methods 232:3–14
Singel KL, Segal BH (2016) NOX2-dependent regulation of inflammation. Clin Sci (Lond) 130:479–490
Lambeth JD (2004) NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol 4:181–189
Kikuchi H, Hikage M, Miyashita H et al (2000) NADPH oxidase subunit, gp91(phox) homologue, preferentially expressed in human colon epithelial cells. Gene 254:237–243
Leoni G, Alam A, Neumann P-A et al (2013) Annexin A1, formyl peptide receptor, and NOX1 orchestrate epithelial repair. J Clin Invest 123:443–454
Jones RM, Luo L, Ardita CS et al (2013) Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated generation of reactive oxygen species. EMBO J 32:3017–3028
Lushchak VI (2014) Free radicals, reactive oxygen species, oxidative stress and its classification. Chem Biol Interact 224:164–175
Go Y-M, Jones DP (2017) Redox theory of aging: implications for health and disease. Clin Sci (Lond) 131:1669–1688
Sies H, Berndt C, Jones DP (2017) Oxidative stress. Annu Rev Biochem 86:715–748
Winterbourn CC, Hampton MB (2008) Thiol chemistry and specificity in redox signaling. Free Radic Biol Med 45:549–561
Schieber M, Chandel NS (2014) ROS function in redox signaling and oxidative stress. Curr Biol 24:R453–R462
Moi P, Chan K, Asunis I et al (1994) Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region. Proc Natl Acad Sci U S A 91:9926–9930
McMahon M, Thomas N, Itoh K et al (2006) Dimerization of substrate adaptors can facilitate cullin-mediated ubiquitylation of proteins by a “tethering” mechanism: a two-site interaction model for the Nrf2-Keap1 complex. J Biol Chem 281:24756–24768
Dinkova-Kostova AT, Holtzclaw WD, Cole RN et al (2002) Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants. Proc Natl Acad Sci U S A 99:11908–11913
Jones RM, Desai C, Darby TM et al (2015) Lactobacilli modulate epithelial cytoprotection through the Nrf2 pathway. Cell Rep 12:1217–1225
Sadlowski CM, Maity S, Kundu K et al (2017) Hydrocyanines: a versatile family of probes for imaging radical oxidants in vitro and in vivo. Mol Sys Des Eng 2:191–200
Kundu K, Knight SF, Willett N et al (2009) Hydrocyanines: a class of fluorescent sensors that can image reactive oxygen species in cell culture, tissue, and in vivo. Angew Chem Int Ed Engl 48:299–303
Ardita CS, Mercante JW, Kwon YM et al (2014) Epithelial adhesion mediated by pilin SpaC is required for Lactobacillus rhamnosus GG-induced cellular responses. Appl Environ Microbiol 80:5068–5077
Alam A, Leoni G, Quiros M et al (2016) The microenvironment of injured murine gut elicits a local pro-restitutive microbiota. Nat Microbiol 1:15021
Pizarro G, Ríos E (2004) How source content determines intracellular Ca2+ release kinetics. Simultaneous measurement of [Ca2+] transients and [H+] displacement in skeletal muscle. J Gen Physiol 124:239–258
Shapovalov SA, Kiseleva YS (2008) The association of cyanine dye cations with phenol red anions in aqueous solutions. Russ J Phys Chem 82:972–977
Zhdanov AV, Aviello G, Knaus UG et al (2017) Cellular ROS imaging with hydro-Cy3 dye is strongly influenced by mitochondrial membrane potential. Biochim Biophys Acta 1861:198–204
Acknowledgments
The authors acknowledge support from the NIH AI64462 and CA179424.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Saeedi, B.J., Chandrasekharan, B., Neish, A.S. (2019). Hydro-Cy3-Mediated Detection of Reactive Oxygen Species In Vitro and In Vivo. In: Knaus, U., Leto, T. (eds) NADPH Oxidases. Methods in Molecular Biology, vol 1982. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9424-3_20
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9424-3_20
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9423-6
Online ISBN: 978-1-4939-9424-3
eBook Packages: Springer Protocols