Imaging Intestinal ROS in Homeostatic Conditions Using L-012

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.