500095 | OxyDNA Assay Kit, Fluorometric

500095
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      Overview

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      Catalogue NumberAvailability Packaging Qty/Pack Price Quantity
      500095-1KIT
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      Limited Availability Limited Availability
      Discontinued
      There is a newer version of this product
      Limited Quantities Available
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          1 kit
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          Description
          Overview Assay kit used to detect oxidative damage to DNA in vitro. Based on the direct binding of a fluorescent probe to the DNA adduct 8-oxoguanine, a major oxidation product and an important indicator of free radical-induced DNA damage and oxidative stress. The FITC-conjugate binds to 8-oxoguanine in damaged cells and fluorescence is monitored using flow cytometery (excitation 495 nm, emission 515 nm).
          Catalogue Number 500095
          Brand Family Calbiochem®
          References
          References Silva, P.F.N. et al. 2007. Theriogenology 67, 609.
          Babbar, N. and Casero, Jr., R.A. 2006. Cancer Res. 66, 11125.
          Lai, K.N., et al. 2006. Nephrol. Dial. Transplant. 21, 1188.
          Sampson, M.J. et al. 2006. Diabetes Care 29, 283.
          Maniscalco, W.M., et al. 2005. Pediatric Res. 58, 594.
          Osella, D., et al. 2005. Inorganica Chimica Acta 358, 1993.
          Adamek B et al. 2004. Polish Journal of Environmental Studies 13 Suppl. II, 29.
          Roper, J.M., et al. 2004. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L1045.
          Ogawa, Y. et al. 2003. International Journal of Molecular Medicine 11, 27.
          Ogawa, Y. et al. 2003. International Journal of Molecular Medicine 11, 149.
          Sparrow J.R. et al. 2003. Investigative Ophthalmology and Visual Science (14)5, 2245.
          Tao Peng, H-M. et al. 2003. World J Gastroenterol 9, 2186.
          Riemschneider, S., et al. 2002. Acta Derm. Venereol. 82, 325.
          de Zwart, L.L., et al. 1999. Free Radic. Biol. Med. 26, 202.
          Tsurudome, Y., et al. 1999. Carcinogenesis 20, 1573.
          Inoue, M., et al. 1998. Jpn. J. Cancer Res. 89, 691.
          Kasai, H. 1997. Mutat. Res. 387, 147.
          Cooke, C., et al. 1996. Toxicol. Ecotoxicol. News 3, 101.



          Selected Citations
          Roper, J. M., et al. 2004. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L1045.
          Babbar, N., and Casero, R. A., Jr., 2006. Cancer Res. 66, 23.


          Product Information
          Detection method Flow cytometry or fluorescence microscopy
          Form 50 Tests
          Kit contains Wash Concentrate, Binding Protein-FITC Conjugate, and a user protocol.
          Applications
          Application References Fluorescent Microscopy Lai, K.N., et al. 2006. Nephrol. Dial. Transplant. 21, 1188. Maniscalco, W.M., et al. 2005. Pediatric Res. 58, 594. Roper, J.M., et al. 2004. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L1045. Other Applications Babbar, N. and Casero, Jr., R.A. 2006. Cancer Res. 66, 11125. Osella, D., et al. 2005. Inorganica Chimica Acta 358, 1993. Riemschneider, S., et al. 2002. Acta Derm. Venereol. 82, 325.
          Biological Information
          Assay time 4 h
          Sample Type Cells
          Physicochemical Information
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          Product Usage Statements
          Intended use The Calbiochem® OxyDNA Kit is an in vitro fluorescent protein binding method for the detection of oxidative damage to DNA in fixed permeabilized cells using flow cytometry or fluorescence microscopy. Material of both human and animal origin can be tested.
          Storage and Shipping Information
          Ship Code Ambient Temperature Only
          Toxicity Multiple Toxicity Values, refer to MSDS
          Storage +2°C to +8°C
          Storage Conditions Upon arrival store the entire contents of the kit at 4°C. Note: FITC conjugate must be stored in the dark.
          Do not freeze No
          Packaging Information
          Transport Information
          Supplemental Information
          Kit contains Wash Concentrate, Binding Protein-FITC Conjugate, and a user protocol.
          Specifications

          Documentation

          MSDS

          Languages
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          Certificates of Analysis

          TitleLot Number
          500095

          References

          Reference overview
          Silva, P.F.N. et al. 2007. Theriogenology 67, 609.
          Babbar, N. and Casero, Jr., R.A. 2006. Cancer Res. 66, 11125.
          Lai, K.N., et al. 2006. Nephrol. Dial. Transplant. 21, 1188.
          Sampson, M.J. et al. 2006. Diabetes Care 29, 283.
          Maniscalco, W.M., et al. 2005. Pediatric Res. 58, 594.
          Osella, D., et al. 2005. Inorganica Chimica Acta 358, 1993.
          Adamek B et al. 2004. Polish Journal of Environmental Studies 13 Suppl. II, 29.
          Roper, J.M., et al. 2004. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L1045.
          Ogawa, Y. et al. 2003. International Journal of Molecular Medicine 11, 27.
          Ogawa, Y. et al. 2003. International Journal of Molecular Medicine 11, 149.
          Sparrow J.R. et al. 2003. Investigative Ophthalmology and Visual Science (14)5, 2245.
          Tao Peng, H-M. et al. 2003. World J Gastroenterol 9, 2186.
          Riemschneider, S., et al. 2002. Acta Derm. Venereol. 82, 325.
          de Zwart, L.L., et al. 1999. Free Radic. Biol. Med. 26, 202.
          Tsurudome, Y., et al. 1999. Carcinogenesis 20, 1573.
          Inoue, M., et al. 1998. Jpn. J. Cancer Res. 89, 691.
          Kasai, H. 1997. Mutat. Res. 387, 147.
          Cooke, C., et al. 1996. Toxicol. Ecotoxicol. News 3, 101.



          Selected Citations
          Roper, J. M., et al. 2004. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L1045.
          Babbar, N., and Casero, R. A., Jr., 2006. Cancer Res. 66, 23.


          User Protocol

          Revision 11-June-2010 JSW
          Form 50 Tests
          Detection method Flow cytometry or fluorescence microscopy
          Storage Upon arrival store the entire contents of the kit at 4°C. Note: FITC conjugate must be stored in the dark.
          Intended use The Calbiochem® OxyDNA Kit is an in vitro fluorescent protein binding method for the detection of oxidative damage to DNA in fixed permeabilized cells using flow cytometry or fluorescence microscopy. Material of both human and animal origin can be tested.
          Background Oxidative injury to macromolecules is indicated in a wide range of pathological conditions. Damage is mediated via free radicals that can be created by a range of agents, e.g. xenobiotics, environmental toxins, smoking, radiation, ischaemia-reperfusion injury oxidising agents and normal or disturbed metabolic activity. These free radicals may react with DNA causing reversible and irreversible damage. This can lead to mutation, carcinogenesis, teratogenesis or cell death. The probe in the OxyDNA kit is specific for 8-oxoguanine. 8-oxoguanine (as part of the oxidized nucleotide 8-oxyguanosine) is formed during free radical damage to DNA and is a sensitive and specific indicator of oxidative DNA damage. 8-oxoguanine is a particularly important biomarker of oxidative DNA damage as it is formed in relatively large quantities and 8-oxoguanine formation can lead to mutations, such as the substitution of thymine for guanine and cytosine for adenine. Previously, 8-oxoguanine was difficult to detect, requiring HPLC analysis, however by utilizing a binding protein with high avidity and specificity for 8-oxoguanine, the OxyDNA kit provides a simple, convenient, sensitive fluorescence method for detecting for oxidative DNA damage.
          Principles of the assay The Calbiochem® OxyDNA Kit utilizes a direct fluorescent protein binding technique. After cells have been fixed and permeabilized, the FITC-Conjugate is added and binds to the 8-oxoguanine moiety present in the 8-oxoguanosine of oxidized DNA. The presence of oxidized DNA is indicated by a green/yellow fluorescence that can be read using flow cytometry or fluorescence microscopy.
          Materials provided • Wash Concentrate (Kit component No. KP10401-55ML): 1 bottle, 55 ml, 25X Tris-buffered Saline/TWEEN® 20 Detergent (TBST), containing thimerosal
          • FITC-Conjugate Concentrate (Kit Component No. KP10402-500UL): 1 vial, 0.5 ml Binding Protein conjugated to Fluorescein, containing thimerosal
          Materials Required but not provided Micropipettes: 20 µl to 100 µl, 200 µl to 1000 µl
          Test tubes
          1 L beaker
          Graduated cylinder
          Reagents for fixing and permeabilising cells
          Deionized/Distilled water
          37°C water bath
          Flow cytometer with appropriate filter combination for FITC (excitation filter 495 nm, barrier filter 515 nm)
          Precautions and recommendations The OxyDNA kit contains a chemical known to the State of California to cause birth defects or other reproductive harm (California Prop 65: thimerosal).
          Dispose of all specimens in accordance with good laboratory practice.
          Wear protective clothing, disposable latex gloves and eye protection while handling specimens and performing the assay. Wash hands thoroughly when finished.
          Do not pipette materials by mouth and never eat or drink at the laboratory workbench.
          Do not mix or substitute reagents from different kit lot numbers.
          Care must be taken not to contaminate components and always use fresh pipette tips for each sample and component.
          Do not use reagents that are cloudy or that have precipitated out of solution. High quality distilled or deionised water is required for the Wash Solution. The use of poor quality or contaminated water may lead to background colour in the assay.
          Allow all reagents to come to room temperature (20-25°C) and mix well prior to use.
          Avoid leaving reagents in direct sunlight and/or above 4°C for extended periods.
          Always use clean, preferably disposable, glassware for all reagent preparation.
          Preparation Live cells should be shielded from high oxygen tensions (e.g. room air) and unnecessary mechanical stress (e.g. mixing and washing) as these can lead to increased oxidative DNA damage and high levels of fluorescence in untreated/control samples. DNA is more stable in fixed cells.
          Reagent preparation • Wash Solution: Dilute the Wash Concentrate 1:25 with diH2O. For example, to prepare 250 ml Wash Solution add 10 ml of Wash Concentrate to 240 ml diH2O. Prepare only the volume of Wash Solution required for the assay. • 1X FITC-Conjugate: Dilute the FITC-Conjugate 1:10 with Wash Solution. For example, to prepare 1 ml 1X FITC-Conjugate add 100 µl FITC-Conjugate to 900 µl Wash Solution. To dispense conjugate, remove cap and pipette directly from the bottle. Keep FITC-Conjugate solutions in the dark when not in use. These dilutions of FITC-Conjugate have been used successfully for a number of cell lines e.g. Hep G2, IMR-32 and CHO, sperm and lymphocytes. In some experimental situations, it may be necessary to optimize the dilution of the conjugate used. This will be dependent on the particular cell type used and the experimental conditions used to induce oxidative damage. If the incorrect concentration of conjugate is used, high background staining and non-specific binding may occur.
          Detailed protocol Assay Protocol for Flow Cytometry Assay

          Oxidative DNA damage can be found in most cell types and can be caused by many different mechanisms. As a result, the protocol outlined below should only be regarded as a guideline and the procedures may need to be modified according to the experimental model or cell type being studied.

          1. Fix and permeabilize the cells to be tested.
          2. Wash with Wash Solution.
          3. Add 100 µl 1X FITC-Conjugate to the cell pellet and incubate in the dark for 60 min at room temperature.
          4. Wash with Wash Solution.
          5. Read fluorescence using a flow cytometer at an excitation wavelength of 495 nm and barrier filter of 515 nm.
          Specificity The Calbiochem® OxyDNA Kit is specific for the 8-oxoguanine moiety of 8-oxoguanosine present in oxidized DNA and shows no measurable cross reactivity with unoxidized guanine / guanosine or other unoxidized nucleotides.
          Application references Fluorescent Microscopy Lai, K.N., et al. 2006. Nephrol. Dial. Transplant. 21, 1188. Maniscalco, W.M., et al. 2005. Pediatric Res. 58, 594. Roper, J.M., et al. 2004. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L1045. Other Applications Babbar, N. and Casero, Jr., R.A. 2006. Cancer Res. 66, 11125. Osella, D., et al. 2005. Inorganica Chimica Acta 358, 1993. Riemschneider, S., et al. 2002. Acta Derm. Venereol. 82, 325.
          Registered Trademarks Calbiochem® is a registered trademark of EMD Chemicals, Inc.
          Tween® is a registered trademark of ICI Americas, Inc.
          Interactive Pathways™ is a trademark of EMD Chemicals, Inc.

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