DHE probe (Intracellular ROS assay) Principle
Cell permeant reagent Dihydroethidium (DHE) is is a fluorogenic dye widely used to detect O2 .− generation in situ, in vitro, and ex vivo. This is because the DHE probe is user-friendly and has high sensitivity. Oxidation of DHE by O2 .− yields 2-hydoxyethidium (2-OH-E+ ). However, other oxidants such as ONOO− , • OH, H2 O2 , compound I, and compound II can produce a 2-electron oxidation product, E+ , with similar fluorescence characteristics.
According to literature, superoxide oxide DHE to form 2-hydroxyethidium (2-OH-E+). Additionally, other sources of Reactive Oxygen Species (ROS) can oxidize DHE, by non-specific oxidation, to form ethidium (E+). The user can monitor their formation by spectrofluorimetry. The conditions are the following: ex 500-530 nm/em 590-620 nm and ex 480 nm/em 576 nm, respectively.
As a result of normal cellular metabolism, living organisms produce ROS. At low to moderate concentrations, they take part in physiological cell processes. However,at high concentrations, they produce adverse modifications to cell components, such as lipids, proteins, and DNA. The shift in balance between oxidant/antioxidant in favor of oxidants is termed “oxidative stress”. Oxidative stress contributes to many pathological conditions. Between them, cancer, neurological disorders, atherosclerosis, hypertension, ischemia/perfusion,diabetes, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease,and asthma can be included.