DCFH-DA probe (Intracellular ROS assay)
Researchers commonly use the DCFH-DA probe for detecting intracellular H2O2 and oxidative stress. This probe is cell-permeable and is hydrolyzed intracellularly to the DCFH carboxylate anion, which is retained in the cell. Two-electron oxidation of DCFH results in the formation of a fluorescent product called dichlorofluorescein (DCF). Consequently, the user can monitor DCF by several fluorescence-based techniques (e.g., confocal microscopy, flow cytometry). This is a relatively easy and user-friendly assay that has become immensely popular. In fact, investigators have routinely used DCFH-DA to measure intracellular generation of H2O2 and other oxidants or monitor redox signaling changes in cells in response to intra- or extracellular activation with oxidative stimulus.
Cell permeant reagent 2’-7’dichlorofluorescin diacetate (DCFH-DA) is a fluorogenic dye that measures hydroxyl, peroxyl and other ROS activity.
After cell uptake, cellular esterases deactylate DCFH-DA, yielding a non-fluorescent compound. Later, ROS oxidize DCFH-DA into 2’-7’dichlorofluorescein (DCF). Then, the user can detect DCF by fluorimetry, flow cytometry or fluorescence microscopy, with a maximum excitation and emission spectra of 495 nm and 529 nm, respectively, as it is a fluorescent compound.
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.