Name: assaying dna damage in hippocampal neurons using the comet assay
Uploaded: 2012-12-19T13:00:00
Description: Watch this Scientific Journal Video about Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay at JoVE.com

This work was supported by the IMPACT Award from the Department of Radiation Oncology, University of Alabama-Birmingham Comprehensive Cancer Center, the Fighting Children's Cancer Foundation, and the Gabrielle's Angel Foundation (to ESY.).

1. Cell Culture
 <ol>
 <li>Culture neuronal cells and treat them as needed.</li>
 <li>Harvest cells into 15 ml tubes: aspirate media, rinse with phosphate buffered saline (PBS, calcium and magnesium free), add trypsin, collect in 15 ml tubes and neutralize trypsin with appropriate serum containing media.</li>
 <li>Spin at 1,000 x g for 5 min.</li>
 <li>Aspirate media.</li>
 <li>Resuspend cells in PBS.</li>
 <li>Spin at 1,000 x g for 5 min.</li>
 <li>Aspirate media and resuspend cells in fresh PBS.</li>
 ...

<ol>
	<li>Olive, P. L., Banath, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay:+a+method+to+measure+DNA+damage+in+individual+cells.">The comet assay: a method to measure DNA damage in individual cells.</a> Nat. Protoc. 1, 23-29 (2006).</li><li>Hovhannisyan, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+in+combination+with+the+comet+assay+and+micronucleus+test+in+genetic+toxicology.">Fluorescence in situ hybridization in combination with the comet assay and micronucleus test in genetic toxicology.</a> Mol. Cytogenet. 3, (2010).</li><li>Olive, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay.+An+overview+of+techniques.">The comet assay. An overview of techniques.</a> Methods Mol. Biol. 203, 179-194 (2002).</li><li>Olive, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+DNA+damage+in+individual+cells+by+analysis+of+histone+H2AX+phosphorylation.">Detection of DNA damage in individual cells by analysis of histone H2AX phosphorylation.</a> Methods Cell Biol. 75, 355-373 (2004).</li><li>Olive, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+the+comet+assay+in+radiobiology.">Impact of the comet assay in radiobiology.</a> Mutat. Res. 681, 13-23 (2009).</li><li>Olive, P. L., Banath, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+H2AX+phosphorylation+after+exposure+to+cisplatin.">Kinetics of H2AX phosphorylation after exposure to cisplatin.</a> Cytometry B Clin. Cytom. 76, 79-90 (2009).</li><li>Olive, P. L., Durand, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+DNA+damage+using+the+comet+assay.">Heterogeneity in DNA damage using the comet assay.</a> Cytometry A. 66, 1-8 (2005).</li><li>Collins, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay+for+DNA+damage+and+repair.">The comet assay for DNA damage and repair.</a> Molecular biotechnology. 26, 249-261 (2004).</li><li>Fairbairn, D. W., Olive, P. L., O'Neill, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay:+a+comprehensive+review.">The comet assay: a comprehensive review.</a> Mutation Research/Reviews in Genetic Toxicology. 339, 37-59 (1995).</li><li>Yang, E. S., Nowsheen, S., Wang, T., Thotala, D. K., Xia, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycogen+synthase+kinase+3B+inhibition+enhances+repair+of+DNA+double-strand+breaks+in+irradiated+hippocampal+neurons.">Glycogen synthase kinase 3B inhibition enhances repair of DNA double-strand breaks in irradiated hippocampal neurons.</a> Neuro-Oncology. 13, 459-470 (2011).</li><li>Yang, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lithium-mediated+protection+of+hippocampal+cells+involves+enhancement+of+DNA-PK+dependent+repair+in+mice.">Lithium-mediated protection of hippocampal cells involves enhancement of DNA-PK dependent repair in mice.</a> The Journal of clinical investigation. , 119-1124 (2009).</li><li>Collins, A. R., Harrington, V., Drew, J., Melvin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+modulation+of+DNA+repair+in+a+human+intervention+study.">Nutritional modulation of DNA repair in a human intervention study.</a> Carcinogenesis. 24, 511-515 (2003).</li><li>Smith, C. C., O'Donovan, M. R., Martin, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hOGG1+recognizes+oxidative+damage+using+the+comet+assay+with+greater+specificity+than+FPG+or+ENDOIII.">hOGG1 recognizes oxidative damage using the comet assay with greater specificity than FPG or ENDOIII.</a> Mutagenesis. 21, 185-190 (2006).</li><li>Rosenquist, T. A., Zharkov, D. O., Grollman, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+characterization+of+a+mammalian+8-oxoguanine+DNA+glycosylase.">Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase.</a> Proceedings of the National Academy of Sciences. 94, 7429-7434 (1997).</li><li>Radicella, J. P., Dherin, C., Desmaze, C., Fox, M. S., Boiteux, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+characterization+of+hOGG1,+a+human+homolog+of+the+OGG1+gene+of+Saccharomyces+cerevisiae.">Cloning and characterization of hOGG1, a human homolog of the OGG1 gene of Saccharomyces cerevisiae.</a> Proceedings of the National Academy of Sciences. 94, 8010-8015 (1997).</li><li>Banath, J. P., Klokov, D., MacPhail, S. H., Banuelos, C. A., Olive, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+gammaH2AX+foci+as+an+indication+of+lethal+DNA+lesions.">Residual gammaH2AX foci as an indication of lethal DNA lesions.</a> BMC Cancer. 10, 4 (2010).</li><li>Banath, J. P., Macphail, S. H., Olive, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+sensitivity,+H2AX+phosphorylation,+and+kinetics+of+repair+of+DNA+strand+breaks+in+irradiated+cervical+cancer+cell+lines.">Radiation sensitivity, H2AX phosphorylation, and kinetics of repair of DNA strand breaks in irradiated cervical cancer cell lines.</a> Cancer Res. 64, 7144-7149 (2004).</li><li>Bonner, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[gamma]H2AX+and+cancer.">[gamma]H2AX and cancer.</a> Nat. Rev. Cancer. 8, 957-967 (2008).</li><li>Finney, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+Field+Gel+Electrophoresis.">Pulsed Field Gel Electrophoresis.</a> Current protocols in molecular biology. , (2000).</li><li>Electrophoresis, F. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pulsed-field gel electrophoresis. , (2000).</li><li>Nowsheen, S., Bonner, J. A., Yang, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+poly+(ADP-Ribose)+polymerase+inhibitor+ABT-888+reduces+radiation-induced+nuclear+EGFR+and+augments+head+and+neck+tumor+response+to+radiotherapy.">The poly (ADP-Ribose) polymerase inhibitor ABT-888 reduces radiation-induced nuclear EGFR and augments head and neck tumor response to radiotherapy.</a> Radiotherapy and Oncology. , (2011).</li></ol>

The comet assay has the unique capacity of analyzing individual cells. This is advantageous in identification of subpopulations of cells that demonstrate differential response to cytotoxic agents. A few practical limitations have to be taken into account. The number of cells that can be evaluated individually may vary depending on the individual. The sample size needs to be increased if there is variance in DNA damage within a population. Viable single-cell suspension is critical for this assay since predominant presence...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>1.5 ml tubes</td>
 <td>Santa Cruz Biotechnology, Inc</td>
 <td>Sc-200271</td>
 <td></td>
 </tr>
 <tr>
 <td>10X TBE (Tris base, boric acid, EDTA)</td>
 <td>Fisher Scientific</td>
 <td>BP13331</td>
 <td></td>
 </tr>
 <tr>
 <td>15 ml tube</td>
 <td>Fisher Scientific</td>
 <td>0553851</td>
 <td></td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Sigma</td>
 <td>A5093</td>
 <td></td>
 </tr>
 <tr>
 <td>Aluminum foil</td>
 <td>Fisher Scientific</td>
 <td>01213101</td>
 <td></td>
 </tr>
 <tr>
 <td>Beaker</td>
 <td>Fisher Scientific</td>
 <td>FB102300</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Thermo Scientific</td>
 <td>75004261PR</td>
 <td></td>
 </tr>
 <tr>
 <td>Comet Assay software</td>
 <td>Comet Assay IV Image Analysis System</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Cylinder</td>
 <td>Fisher Scientific</td>
 <td>08555F</td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>GIBCO</td>
 <td>15575</td>
 <td></td>
 </tr>
 <tr>
 <td>Electrophoresis chamber</td>
 <td>Thermo Scientific</td>
 <td>09528101</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Fisher Scientific</td>
 <td>A407P4</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluorescent microscope</td>
 <td>Zeiss Axio Vision</td>
 <td></td>
 <td>Any fluorescent microscope with green filter will suffice</td>
 </tr>
 <tr>
 <td>Hemacytometer</td>
 <td>Fisher Scientific</td>
 <td>0267152</td>
 <td></td>
 </tr>
 <tr>
 <td>Low melting point agarose</td>
 <td>Promega</td>
 <td>V2111</td>
 <td></td>
 </tr>
 <tr>
 <td>Microwave</td>
 <td>Sears</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate buffered saline, calcium free, magnesium free</td>
 <td>HyClone</td>
 <td>SH3025601</td>
 <td></td>
 </tr>
 <tr>
 <td>Power supply</td>
 <td>BioRad</td>
 <td>1645050</td>
 <td></td>
 </tr>
 <tr>
 <td>Refrigerator</td>
 <td>Sears</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ruler</td>
 <td>Staples</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Slide</td>
 <td>Fisher Scientific</td>
 <td>12550143</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium chloride</td>
 <td>Sigma</td>
 <td>S7653</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium hydroxide</td>
 <td>Sigma</td>
 <td>S5881</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium lauryl sarcosinate</td>
 <td>Fisher Scientific</td>
 <td>S529</td>
 <td></td>
 </tr>
 <tr>
 <td>Sybr green</td>
 <td>Invitrogen</td>
 <td>S7585</td>
 <td></td>
 </tr>
 <tr>
 <td>Tray</td>
 <td>Fisher Scientific</td>
 <td>15242B</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>Sigma</td>
 <td>T6066</td>
 <td></td>
 </tr>
 <tr>
 <td>Triton-X 100</td>
 <td>Sigma</td>
 <td>T8787</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>HyClone</td>
 <td>SH3023601</td>
 <td></td>
 </tr>
 <tr>
 <td>Vortex</td>
 <td>Fisher Scientific</td>
 <td>02216108</td>
 <td></td>
 </tr>
 <tr>
 <td>Water bath</td>
 <td>Fisher Scientific</td>
 <td>154622Q</td>
 <td></td>
 </tr>
 </tbody>
 </table>

assaying dna damage in hippocampal neurons using the comet assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

Watch this Scientific Journal Video about Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay at JoVE.com

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Research

JoVE Journal

Neuroscience

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...