title: Establishing the subcellular localization of photodynamically-induced ROS using 3,3'-diaminobenzidine: A methodological proposal, with a proof-of-concept demonstration.
abstract: The critical involvement of reactive oxygen species (ROS) in both physiological and pathological processes in cell biology makes their detection and assessment a fundamental topic in biomedical research. Established methodologies to study ROS in cell biology take advantage of oxidation reactions between the ROS and a reduced probe. After reacting the probe reveals the presence of ROS either by the appearance of colour (chromogenic reaction) or fluorescence (fluorogenic reaction). However current methodologies rarely allow for a site-specific detection of ROS production. Here we propose a colorimetric reaction driven by the oxidation of 3,3'-diaminobenzidine (DAB) by photodynamically-produced ROS that allows for fine detection of the ROS production site. The introduced methodology is fast, easy to implement and permits cellular resolution at the submicrometric level. Although the basic protocol is proved in a photodynamic model of ROS generation, the principle is applicable to many different scenarios of intracellular ROS production. As a consequence this proposed methodology should greatly complement other techniques aiming at establishing a precise subcellular localization of ROS generation.
authors.affiliation: Department of Biology, Faculty of Sciences, Autonomous University of Madrid, Spain; Institute of Research and Technology in Animal Reproduction, Faculty of Veterinary Sciences, University of Buenos Aires, Argentina. Electronic address: juancarlos.stockert@gmail.com.
authors.forename: Juan C
authors.initials: JC
authors.lastname: Stockert
authors.affiliation: AIAS, Aarhus Institute of Advanced Studies, Aarhus University, Denmark.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Blázquez-Castro
mesh_terms: D015100:3,3'-Diaminobenzidine / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry* / Q000528:radiation effects; D008027:Light; D008967:Molecular Biology / Q000379:methods*; D010084:Oxidation-Reduction / Q000528:radiation effects; D017382:Reactive Oxygen Species / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D017382:Reactive Oxygen Species; D015100:3,3'-Diaminobenzidine
keywords: 3,3′-Diaminobenzidine; Colorimetric reaction; Localized ROS generation; Photoactive dyes; Photosensitizers; ROS