Bestellinformationen
Produktabbildungen
MnSOD dose responses of A549, HeLa and HepG2 cells to toxic stresses.
A549 or HeLa cells were plated at 18,000 cells/cm2 (37,000 cells/cm2 for H...
Phospho-histone H2A.X dose responses of A549, HeLa and HepG2 cells to toxic stresses.
A549 or HeLa cells were plated at 18,000 cells/cm2 (37,000...
)
)
)
)
Manganese Superoxide Dismutase (MnSOD) and Histone H2A.X Phosphorylation Assay
Description:
Manganese Superoxide Dismutase (MnSOD) and Histone H2A.X Phosphorylation Assay
Product Overview:
Millipore’s HCS233 assay provides a complete solution for identifying and quantifying manganese superoxide dismutase (MnSOD) and phospho-histone H2A.X(Ser139) in cellular imaging studies. The reagents in the kit have been specifically optimized for HCS applications. The assay is designed to enable visualization and quantitative detection of MnSOD and phosphorylated histone H2A.X, allowing the characterization of the oxidative stress response, genotoxicity, and screening of compounds that may induce, inhibit or repair cellular injury such as DNA damage, oxidative stress and inflammation. The nuclear dye (Hoechst 33342) may be used for measurements of cell number, DNA content and nuclear size. Additionally, the assay can be multiplexed with other probes, e.g., for apoptotic pathway studies, oncology drug efficacy trials or in vitro toxicology applications.
Background Information:
Detection and quantification of phospho-histone H2A.X (Ser139) foci as an indicator of oxidative stress, DNA damage, repair and apoptosis is of great interest to researchers investigating these pathways. Additionally, recent data indicates that phospho-histone H2A.X (Ser139) may have additional cellular functions which have yet to be fully elucidated, representing a focus of ongoing research. High Content Screening technology represents an ideal tool for performing quantitative analysis of phospho-histone H2A.X (Ser139) expression.
SOD
Superoxide dismutase (SOD) is an antioxidant enzyme involved in the defense system against reactive oxygen species (ROS). SOD catalyzes the dismutation reaction of superoxide radical anion (O2-) to hydrogen peroxide, which is then catalyzed to innocuous O2 and H2O by glutathione peroxidase and catalase. Several classes of SOD have been identified. These include intracellular copper, zinc SOD (Cu, Zn SOD/SOD1), mitochondrial manganese SOD (Mn SOD/SOD2) and extracellular Cu, Zn SOD (EC SOD/SOD3). The manganese containing 80 kDa tetrameric enzyme SOD2 is located in the mitochondrial matrix in close proximity to a primary endogenous source of superoxide, the mitochondrial respiratory chain.
H2A.X
One of the principal responses to DNA damage resulting in double-stranded DNA breaks (DSBs) is the activation of the ATM-initiated signaling cascade to arrest cell division until repairs can be made. Agents that cause DSBs include oxidative stress, ionizing radiation, topoisomerase inhibitors, and DNA binding drugs. A major substrate of the kinases in this cascade is H2A.X. H2A.X is a variant isoform of the histone H2A protein, containing several serine residues (including serine 139) in its unique carboxy-terminal amino acid sequence that are rapidly phosphorylated in response to DSBs. H2A.X is rapidly phosphorylated at serine 139 by the ATM kinase in response to even a few DSBs, and accumulates at the sites of DNA damage forming distinct nuclear foci. These events are important for recruitment and maintenance of the DNA repair machinery to the site of the break. Thus the presence of phospho-histone H2A.X (Ser139) is a reliable marker of DSBs and DNA damage. As DNA repair progresses, the number of foci declines.
SOD
Superoxide dismutase (SOD) is an antioxidant enzyme involved in the defense system against reactive oxygen species (ROS). SOD catalyzes the dismutation reaction of superoxide radical anion (O2-) to hydrogen peroxide, which is then catalyzed to innocuous O2 and H2O by glutathione peroxidase and catalase. Several classes of SOD have been identified. These include intracellular copper, zinc SOD (Cu, Zn SOD/SOD1), mitochondrial manganese SOD (Mn SOD/SOD2) and extracellular Cu, Zn SOD (EC SOD/SOD3). The manganese containing 80 kDa tetrameric enzyme SOD2 is located in the mitochondrial matrix in close proximity to a primary endogenous source of superoxide, the mitochondrial respiratory chain.
H2A.X
One of the principal responses to DNA damage resulting in double-stranded DNA breaks (DSBs) is the activation of the ATM-initiated signaling cascade to arrest cell division until repairs can be made. Agents that cause DSBs include oxidative stress, ionizing radiation, topoisomerase inhibitors, and DNA binding drugs. A major substrate of the kinases in this cascade is H2A.X. H2A.X is a variant isoform of the histone H2A protein, containing several serine residues (including serine 139) in its unique carboxy-terminal amino acid sequence that are rapidly phosphorylated in response to DSBs. H2A.X is rapidly phosphorylated at serine 139 by the ATM kinase in response to even a few DSBs, and accumulates at the sites of DNA damage forming distinct nuclear foci. These events are important for recruitment and maintenance of the DNA repair machinery to the site of the break. Thus the presence of phospho-histone H2A.X (Ser139) is a reliable marker of DSBs and DNA damage. As DNA repair progresses, the number of foci declines.
Key Applications:
- Immunocytochemistry
- Immunofluorescence
- in vitro Toxicology Assays
- High Content Analysis
Application Notes:
Oxidative Stress
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Kit or Assay Type:
- High-Content Screening
- High-Content Analysis
Configuration:
Sufficient reagents for 5 X 96 well plates
Components:
- 1. Rabbit Anti-Manganese Superoxide Dismutase (MnSOD) HCS Primary Antibody, 100X
- 2. HCS Secondary Antibody (donkey anti-rabbit IgG, Cy3 conjugate), 200X
- 3. Mouse Anti-Phospho-Histone H2A.X(Ser139) HCS Primary Antibody, 100X
- 4. HCS Secondary Antibody (donkey anti-mouse IgG, FITC conjugate), 200X
- 5. Hoechst HCS Nuclear Stain, 200X
- 6. HCS Fixation Solution with Phenol Red, 2X
- 7. HCS Immunofluorescence Buffer, 1X
- 8. HCS Wash Buffer, 1X
- 9. Etoposide (S/G2 Arrest Control Compound), 250X
- 10. Camptothecin (Control Compound), 250X
- 11. DMSO for Compound Serial Dilution
- 12. Compound Dilution Buffer
- 13. Plate Sealers
Storage Conditions:
Store kit components under the conditions indicated on the labels. Discard any remaining reagents after 6 months.
Detection Methods:
Fluorescent
Antibody Category:
Neuroscience
Materials Required but Not Delivered:
1. Sterile, tissue culture-treated black/clear bottom microplates suitable for High-Content Imaging.
2. Cell-type for assay, e.g., HeLa (human cervical adenocarcinoma, ATCC #CCL-2) or A549 (human lung carcinoma, ATCC #CCL-185) or HepG2 (human hepatocellular carcinoma, ATCC #HB-8065).
3. Tissue culture instruments/supplies (including 37ºC incubator, growth media, flasks/plates, detachment buffer, etc.) for cell type of interest.
4. HCS imaging/analysis system, e.g., GE Healthcare IN Cell Analyzer 1000 with Investigator software. Imaging system must be equipped with beam-splitters and filters capable of reading emission spectra in the blue and red ranges.
2. Cell-type for assay, e.g., HeLa (human cervical adenocarcinoma, ATCC #CCL-2) or A549 (human lung carcinoma, ATCC #CCL-185) or HepG2 (human hepatocellular carcinoma, ATCC #HB-8065).
3. Tissue culture instruments/supplies (including 37ºC incubator, growth media, flasks/plates, detachment buffer, etc.) for cell type of interest.
4. HCS imaging/analysis system, e.g., GE Healthcare IN Cell Analyzer 1000 with Investigator software. Imaging system must be equipped with beam-splitters and filters capable of reading emission spectra in the blue and red ranges.