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NADPH Oxidases pp 313-327 | Cite as

Imaging Intestinal ROS in Homeostatic Conditions Using L-012

  • Emer Conroy
  • Gabriella AvielloEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1982)

Abstract

Reactive oxygen species (ROS) are critical redox regulators of cellular dynamics controlling homeostasis. Although numerous fluorescent probes are currently available to measure ROS in cell-based assays, the short-lived nature of these molecules renders their detection challenging in more complex biological systems, such as the gastrointestinal tract in vivo. However, in the past decade, significant progress has been made in the development of novel imaging technologies and probes, facilitating ROS quantification with high sensitivity, selectivity, and temporal resolution. The IVIS Spectrum (PerkinElmer) is an optical imaging system for small animal imaging allowing precise and noninvasive visualization of fluorescent or bioluminescent signals. Here, we describe a reproducible and comprehensive method for the measurement of physiological intestinal NADPH oxidase-derived ROS by using the chemiluminescent probe L-012. Using transgenic mice deficient in Nox isoforms expressed in the intestinal mucosa, we delineate the contribution of gut epithelial versus immune cell NADPH oxidase activity in homeostatic conditions. We also discuss L-012 probe specificity and potential alternatives for in vivo studies.

Key words

L-012 IVIS NADPH oxidase NOX1 NOX2 p22phox Intestine Microbiota Inflammatory bowel disease 

Notes

Acknowledgments

The authors thank Prof. Ulla G. Knaus for fruitful discussions and Dr. Jacek Zielonka for critical reading of the manuscript. EC is supported by the Irish Cancer Society Collaborative Cancer Research Centre BREAST-PREDICT. GA is supported by the European Crohn’s and Colitis Organisation (ECCO) and the Medical Research Council (MRC).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Conway Institute, University College DublinDublinIreland
  2. 2.The Rowett Institute, University of AberdeenAberdeenUK

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