Constant generation of low levels of reactive oxygen species (ROS) and free radicals is a basic feature of all living cells. Low levels of ROS play an essential role in signaling pathways, whereas increased under oxidative stress, ROS activity result in damage to nucleic acids, proteins and membrane lipids. Accumulation of ROS during oxidative stress is also associated with aging, apoptosis or necrosis, and is implicated in pathological conditions such as; vascular diseases, diabetes, renal ischemia, arteriosclerosis, pulmonary disorders, inflammatory diseases, and cancer. Cellular activity of ROS is offset by antioxidants, numerous repair systems, and replacement of damaged DNA. Probes for measuring intracellular ROS levels provide important tools to study oxidative stress inducers and effects of antioxidant therapies. Assay Genie's ROS Detection Assay Kit is designed for detection of hydroxyl, peroxyl, or other reactive oxygen species in live cells. We utilize H2DCFDA, a unique cell-permeable fluorogenic probe, compatible with phenol red, FBS and BSA to detect reactive oxygen species in live cells. Upon the cell entry, H2DCFDA is modified by cellular esterases to form a non-fluorescent H2DCF. Oxidation of H2DCF by intracellular ROS yields highly a fluorescent product that can be detected by FACS, microplate reader, or fluorescence microscope (Ex/Em 495/529 nm). The fluorescence intensity is proportional to the ROS levels. Our kit provides a simple and specific assay for the real-time measurement of global levels of ROS in living cells. We include sufficient reagents to perform 100 assays and a common ROS inducer as a control for measurement of ROS levels or antioxidant activity with high sensitivity, specificity and accuracy.

Figure: Analysis of oxidative stress in live cells based on ROS staining. Live cells labeled and treated with ROS Inducer and 100 µM of H₂O₂ according to the kit protocol. A. 2 x10 4 Jurkat cells per condition analyzed by FACS. Plotted intensity values show significant increase in ROS production between treatments and controls. B. 1x10 5 Jurkat cells analyzed on a plate reader. Mean+/- standard deviation plotted for 3 replicates per condition. C. Profiling of ROS formation by Fluorescence Microscopy in Jurkat and MCF-7 live cells.