The kit contains a polyclonal rabbit anti-MnSOD antibody, a mouse monoclonal anti-phospho-H2AX antibody, secondary antibodies conjugated to a DyLight™ Fluorophore and various other reagents and buffers required for immunofluorescent detection of MnSOD and phospho-H2AX for high-content screening (HCS) assays.
Reactive oxygen species (ROS) are formed when oxygen gains one or more electrons from a leaky electron transport chain. Production of these species results from normal aerobic respiration and exposure to toxic drugs or UV irradiation. If allowed to accumulate, ROS, including superoxide (O2·-), hydrogen peroxide (H2O2), hydroxyl radical (OH·) and peroxynitrite (ONOO-), disrupt metabolism and damage DNA and proteins. Oxidative DNA damage is difficult to measure when trying to measure both the ROS generated and the resulting DNA damage lesions. This kit uses MnSOD, a mitochondrial-associated SOD induced upon superoxide production. MnSOD uses redox active manganese in its active site to dismute O2·- to H2O2. MnSOD induction indicates recent ROS production, and phospho-H2AX indicates DNA damage. Cumulative oxidative stress and DNA damage is associated with aging, cancer, rheumatoid arthritis and Alzheimer’s disease.
To induce MnSOD and phospho-H2AX activation in RAW 264.7 macrophages, cells were treated overnight with paraquat and iron. Paraquat treatment enables superoxide generation, and iron promotes production of the hydroxyl radical via the Fenton reaction to directly damage DNA. MnSOD is induced in the cytoplasm and H2AX is phosphorylated in the nucleus. The assay was optimized with the Thermo Scientific ArrayScan HCS Reader using the Compartmental Analysis BioApplication Software Module. MnSOD is quantitated using cytoplasmic intensity or the increase in cytoplasmic spot intensity. Phospho-H2AX is quantitated using the total or average nuclear intensity. Cells stained using this kit also can be imaged using fluorescence or confocal microscopy.
Figure 2. Treatment with paraquat and iron induces MnSOD and phosphorylation of H2AX in RAW 264.7 macrophages. Cells were treated for 20 hours with paraquat (500 µM) and iron (200 µg/ml). Cells were fixed and stained according to the kit protocol. Hoechst 33342 dye was used for nuclear staining. Stimulation (bottom panels) induces MnSOD (increased cytosolic intensity) and phosphorylation of H2AX (increased nuclear intensity).
Figure 3. Dose response curves of MnSOD in RAW 264.7 cells treated with paraquat and iron. Cells were treated for 18 hours. Each data point represents eight wells. Curves separate data from three replicate plates.
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