The kit contains a polyclonal rabbit MnSOD antibody, a goat anti-rabbit secondary antibody conjugated to DyLight™ 549 Fluorophore, and various other reagents and buffers required for immunofluorescent detection of MnSOD 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, these ROS, including superoxide (O2-), hydrogen peroxide (H2O2 ), hydroxyl radical (OH·), and peroxynitrite (ONOO-), disrupt metabolism and damage DNA and proteins. To reduce the accumulation of these species, cells induce cellular enzymes, such as SOD and catalase. There are two mammalian SOD isoforms, a mitochondrial-associated SOD, with manganese in its active site, and a cytosolic SOD, with copper and zinc in the active site. The dismutation reaction occurs via the redox metal in the active site. Cumulative oxidative stress has been associated with aging, cancer, rheumatoid arthritis and Alzheimer’s disease.
To induce MnSOD in the cytoplasm of RAW 264.7 macrophages, cells were treated overnight with the superoxide generator, paraquat and iron. The MnSOD Induction Kit assay was optimized with the Thermo Scientific ArrayScan HCS Reader using the Compartmental Analysis BioApplication Software Module. MnSOD can be quantitated using the cytoplasmic intensity or the increase in cytoplasmic spot intensity. Stained cells also can be imaged using fluorescence or confocal microscopy.
Figure 2. Treatment with paraquat and iron induces MnSOD in RAW 264.7 macrophages. After treatment for 20 hours with paraquat (500 µM) and iron (200 µg/ml), cells were fixed and stained according to the kit protocol and imaged using the ArrayScan VTI HCS Reader. Top panels are non-treated cells.
Figure 3. Dose response curves of MnSOD in RAW 264.7 cells treated with paraquat, iron and rotenone. Cells were treated for 18 hours with paraquat and iron or 17 hours with rotenone. Data points represent eight wells. Curves in top panel represent separate data from three replicate plates. Single curve in bottom panel represents data from a single plate.
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