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

这项工作是支持的，战斗的儿童癌症基金会，阿拉巴马州伯明翰大学综合癌症中心的放射肿瘤学，Gabrielle的天使基金（ESY。）系的影响力大奖。

1。细胞培养<ol><li>的文化神经细胞和治疗自己的需要。 </li><li>收获细胞成15毫升管抽吸介质，清洗用磷酸盐缓冲液（PBS，钙和镁免费的），加入胰蛋白酶，收集在15毫升的试管中胰蛋白酶与适当的含药血清媒体。 </li><li>附带在1,000 xg离心5分钟。 </li><li>吸介质。 </li><li>在PBS中重悬细胞。 </li><li>附带在1,000 xg离心5分钟。 </li><li>吸媒体和新鲜的PBS重悬细胞。 </li><li>使用血球计数器或您首选的细胞?...

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	<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. 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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. 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彗星试验分析单个细胞具有独特的能力。这是有利的鉴定表明微分响应的细胞毒性剂的细胞亚群。一个必须考虑一些实际的限制。的细胞数，可以单独评估，可能会有所不同，这取决于个人。样本大小需要增加内的人口在DNA损伤，如果有方差。可行的单细胞悬液，此测定是至关重要的，因为优势坏死或凋亡的细胞的存在下，将加至错误。的细胞凋亡，小的DNA片段（小于50KB），线粒体DNA的裂解和电...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录号 </td><td> 评论（可选） </td></tr><tr><td> 1.5 ml离心管</td><td>圣克鲁斯生物技术公司</td><td> SC-200271 </td><td></td></tr><tr><td> 10X TBE（Tris碱，硼酸，EDTA） </td><td> Fisher Scientific则</td><td> BP13331 </td><td></td></tr><tr><td> 15 ml管</td><td> Fisher Scientific则</td><td> 0553851 </td><td></td></tr><tr><td>琼脂糖</td><td>西格玛</td><td> A5093 </td><td></td></tr><tr><td>铝箔</td><td> Fisher Scientific则</td><td> 01213101 </td><td></td></tr><tr><td>烧杯</td><td> Fisher Scientific则</td><td> FB102300 </td><td></td></tr><tr><td>离心分离</td><td> Thermo Scientific的</td><td> 75004261PR </td><td></td></tr><tr><td>彗星试验检测软件</td><td>彗星试验IV图像分析系统</td><td></td><td></td></tr><tr><td>气缸</td><td> Fisher Scientific则</td><td> 08555F </td><td></td></tr><tr><td> EDTA </td><td> GIBCO </td><td> 15575 </td><td></td></tr><tr><td>电泳室</td><td> Thermo Scientific的</td><td> 09528101 </td><td></td></tr><tr><td>乙醇</td><td> Fisher Scientific则</td><td> A407P4 </td><td></td></tr><tr><td>荧光显微镜</td><td>蔡司Axio的愿景</td><td></td><td>荧光显微镜绿色过滤器就足够了</td></tr><tr><td>血球</td><td> Fisher Scientific则</td><td> 0267152 </td><td></td></tr><tr><td>低熔点琼脂糖</td><td> Promega公司</td><td> V2111 </td><td></td></tr><tr><td>微波</td><td>西尔斯</td><td></td><td></td></tr><tr><td>磷酸盐缓冲盐水，无钙，镁</td><td>胎牛血清</td><td> SH3025601 </td><td></td></tr><tr><td>电源</td><td> BioRad公司</td><td> 1645050 </td><td></td></tr><tr><td>冰箱</td><td>西尔斯</td><td></td><td></td></tr><tr><td>统治者</td><td>斯台普斯</td><td></td><td></td></tr><tr><td>滑动</td><td> Fisher Scientific则</td><td> 12550143 </td><td></td></tr><tr><td>氯化钠</td><td>西格玛</td><td> S7653 </td><td></td></tr><tr><td>氢氧化钠</td><td>西格玛</td><td> S5881 </td><td></td></tr><tr><td>月桂基肌氨酸钠</td><td> Fisher Scientific则</td><td> S529 </td><td></td></tr><tr><td> SYBR绿色</td><td> Invitrogen公司</td><td> S7585 </td><td></td></tr><tr><td>托盘</td><td>金融机构她的科学</td><td> 15242B </td><td></td></tr><tr><td>三基地</td><td>西格玛</td><td> T6066 </td><td></td></tr><tr><td>的Triton-X 100的</td><td>西格玛</td><td> T8787 </td><td></td></tr><tr><td>胰蛋白酶</td><td>胎牛血清</td><td> SH3023601 </td><td></td></tr><tr><td>涡流</td><td> Fisher Scientific则</td><td> 02216108 </td><td></td></tr><tr><td>水浴</td><td> Fisher Scientific则</td><td> 154622Q </td><td></td></tr></tbody></table>

assaying dna damage in hippocampal neurons using the comet assay

一些药物的目标是DNA修复途径，通过建立DNA损伤诱导细胞死亡。因此，直接测量DNA损伤的进程已经得到了广泛的评估。传统的方法是费时的，昂贵的，资源密集型的时间，需要复制的细胞。与此相反，彗星试验，单细胞凝胶电泳法，是更快的，非侵入性，价格低廉，直接和敏感的DNA损伤和修复措施。所有形式的DNA损伤以及DNA修复可以使用这个强大的技术在单细胞水平的可视化。 彗星试验的原则是高度有序的，完整的DNA，而DNA损伤，破坏了这种组织。受损的DNA的琼脂糖基质渗入和电场进行时，带负电荷的DNA是带正电的向阴极迁移。大的未损坏的DNA链是不能够迁移远离原子核。 DNA损伤创建更小的DNA片段更远较完整的DNA。彗星试验检测，图像分析软件，措施和比较的整体在细胞核中的DNA的荧光强度与已迁移出细胞核的DNA。从迁移的DNA的荧光信号是正比于DNA损伤。更长的明亮的DNA尾着增加DNA损伤。某些测量的参数，该参数是尾矩，它是衡量两者的DNA的量和分布在尾部，尾部长度的DNA和DNA的百分比在尾部。此法可以测量DNA修复以及修复已经发生DNA损伤的决议表示。灵敏度的极限是约50的链断裂每二倍体哺乳动物细胞1,2。与任何DNA损伤剂，如鬼臼乙叉甙处理的细胞，也可以使用作为阳性对照。因此，彗星试验是一种快速，有效的方法来衡量DNA损伤。

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

彗星试验是检测单和双链断裂，包括碱不稳定的的网站和DNA-DNA/DNA-protein交联在所有细胞中的DNA，包括海马神经元的一种有效方式。由于DNA损伤的量的差异，在电场中的方法，利用差DNA迁移。

彗星试验测定DNA损伤的海马神经元

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

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

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

科研

神经科学

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

神经调节和线粒体运输：在较长的时间里，海马神经元的实时成像

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

从产前小鼠海马神经元的分离和培养

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

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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

检测蛋白质棕榈酰化培养海马神经元通过免疫沉淀和酰基-生物素交易所（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

原代海马神经元的磷酸钙转染

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

成像大鼠原代海马神经元树突棘使用结构照明显微镜

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

亚细胞钙荧光和生物发光成像<sup&gt; 2+</sup&gt;老年海马神经元

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

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

条纹分析来研究蛋白质底物使用游离海马神经元的吸引力或令人反感的活动

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

使用高尔基Cox方法评估老年小鼠海马树突状复杂性

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...