Cellomics HCS Reagent Kit for measuring oxidative DNA damage as manganese superoxide dismutase (MnSOD) and phospho-histone 2AX induction.

DNA Content	Phospho-H2AX	MnSOD
Measure three parameters in the same cells			Determine dose responses

Description

The Cellomics MnSOD and Phospho-H2AX Induction Kit measures the production of oxidative DNA damage using manganese superoxide dismutase (MnSOD) and the DNA damage sensor, phospho-histone 2AX. 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 (Figure 1). 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 1. 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 2. 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.

Related Poster Presentations

Kay Opperman, Bhaskar S. Mandavilli, and Richik N. Ghosh (2008) Assessment of Oxidative Stress and DNA Damage in Individual Cells Using Automated, Quantitative Cell-Based Imaging.

Kit Contents

Ordering Information

Screening size kits and components (bulk and custom) available with special pricing. Please call.

To order Cellomics HCS Reagent Kits, please call 800-874-3723 in the U.S.
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