Name: evaluating in vitro dna damage using comet assay
Uploaded: 2017-10-11T14:00:00
Description: Watch this Scientific Journal Video about Evaluating In Vitro DNA Damage Using Comet Assay at JoVE.com

这项研究得到了 NIH、癌症研究所和 CCR 的校内研究项目的支持。所有的作者都获得了来自 NIH, 癌症研究所和 CCR 的内部研究补助金。

1. 准备试剂 <ol> <li> 1x pbs <ol> <li> 用900毫升 dH 2 O 稀释100毫升 10x pbs, 并使用 ph 计将 ph 值调整为7.4。储存在室温下. </li>
	</ol> </li> <li> 溶解解决方案 (LS) <ol> <li> 准备2.5 米氯化钠, 100 毫米二钠 EDTA, 10 毫米三碱, 200 毫米氢氧化钠在900毫升 dH 2 O; 它通常需要大约20分钟, 使混合物完全溶解。使用 ph 计将 ph 值调整到10。加入1% 的十二烷基酸和1% 的海卫 X-100,...

<ol>
	<li>Ostling, O., Johanson, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectrophoretic+study+of+radiation-induced+DNA+damages+in+individual+mammalian+cells.">Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells.</a> Biochem Biophys Res Commun. 123 (1), 291-298 (1984).</li><li>Singh, N. P., McCoy, M. T., Tice, R. R., Schneider, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+quantitation+of+low+levels+of+DNA+damage+in+individual+cells.">A simple technique for quantitation of low levels of DNA damage in individual cells.</a> Exp Cell Res. 175 (1), 184-191 (1988).</li><li>Tice, R. R., 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=Single+cell+gel/comet+assay:+guidelines+for+in+vitro+and+in+vivo+genetic+toxicology+testing.">Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.</a> Environ Mol Mutagen. 35 (3), 206-221 (2000).</li><li>Shah, A. J., Lakkad, B. C., Rao, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotoxicity+in+lead+treated+human+lymphocytes+evaluated+by+micronucleus+and+comet+assays.">Genotoxicity in lead treated human lymphocytes evaluated by micronucleus and comet assays.</a> Indian J Exp Biol. 54 (8), 502-508 (2016).</li><li>Azqueta, A., 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+essential+comet+assay:+a+comprehensive+guide+to+measuring+DNA+damage+and+repair.">The essential comet assay: a comprehensive guide to measuring DNA damage and repair.</a> Arch Toxicol. 87 (6), 949-968 (2013).</li><li>Goldstein, M., Kastan, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+damage+response:+implications+for+tumor+responses+to+radiation+and+chemotherapy.">The DNA damage response: implications for tumor responses to radiation and chemotherapy.</a> Annu Rev Med. 66, 129-143 (2015).</li><li>Gavande, N. 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=DNA+repair+targeted+therapy:+The+past+or+future+of+cancer+treatment?.">DNA repair targeted therapy: The past or future of cancer treatment?.</a> Pharmacol Ther. 160, 65-83 (2016).</li><li>Torgovnick, A., Schumacher, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+mechanisms+in+cancer+development+and+therapy.">DNA repair mechanisms in cancer development and therapy.</a> Front Genet. 6, 157 (2015).</li><li>Weston, V. J., 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=The+PARP+inhibitor+olaparib+induces+significant+killing+of+ATM-deficient+lymphoid+tumor+cells+in+vitro+and+in+vivo.">The PARP inhibitor olaparib induces significant killing of ATM-deficient lymphoid tumor cells in vitro and in vivo.</a> Blood. 116 (22), 4578-4587 (2010).</li><li>Brown, J. S., O'Carrigan, B., Jackson, S. P., Yap, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+DNA+Repair+in+Cancer:+Beyond+PARP+Inhibitors.">Targeting DNA Repair in Cancer: Beyond PARP Inhibitors.</a> Cancer Discov. 7 (1), 20-37 (2017).</li><li>Lu, Y., 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=Chemosensitivity+of+IDH1-Mutated+Gliomas+Due+to+an+Impairment+in+PARP1-Mediated+DNA+Repair.">Chemosensitivity of IDH1-Mutated Gliomas Due to an Impairment in PARP1-Mediated DNA Repair.</a> Cancer Res. 77 (7), 1709-1718 (2017).</li><li>Konca, K., 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+cross-platform+public+domain+PC+image-analysis+program+for+the+comet+assay.">A cross-platform public domain PC image-analysis program for the comet assay.</a> Mutat Res. 534 (1-2), 15-20 (2003).</li><li>Valverde, M., Rojas, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+and+occupational+biomonitoring+using+the+Comet+assay.">Environmental and occupational biomonitoring using the Comet assay.</a> Mutat Res. 681 (1), 93-109 (2009).</li><li>Collins, A. 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彗星试验是一个有效的工具, 以测量单和双 DNA 断裂的细胞水平。该法在遗传毒性和生物13的研究中被广泛应用为 "黄金标准", 从基底病变、DNA 通道、药物开发和碱敏部位不等。在本研究中, 我们分别显示了两个不同的 step-by 步骤的碱性和中性彗星分析的协议。结合单细胞电泳, 荧光显微镜和图像解释, 无论是碱性和中性的彗星检测提供定量的方法来评估的程度 DNA 损伤在体外</em...

彗星试验是第一次开发的 Ostling 和艾里克·乔纳森在1984年通过证明 DNA 片段从细胞核迁移在一个中性条件下1。该技术后来由辛格的et al., 表明碱性条件极大地提高了检测的特异性和重现性的分析2。从那时起, 中性彗星检测主要用于检测双的 dna 断裂, 而碱性彗星检测对较小数量的 dna 损伤更敏感, 包括单和双链 dna 断裂、碱不稳定部位、dna dna 或dna 蛋白交联, 与不完全切除修复相关的 dna 单断裂3,4。这两种方法都允许对片段 dna 进行可视化, 并提供一种直接的方式来定量评估 dna 损伤。彗星试验被认为是一种敏感的方法,体外和体内遗传毒理学研究, 并适用于不同的研究领域, 如早期药物候选选择, 环境监测, 人类生物,以及 DNA 损伤和修复的基础研究5。
该法的原理是, 在电场下, 碎片 dna 从核体 (也称为 "彗星头") 迁移出来, 并在琼脂糖凝胶 (也称为 "彗星尾") 中形成 dna 染色。通过对这种单细胞电泳所形成的 "彗星" 进行分析, 可以量化 DNA 损伤的程度。尾力矩的计算可以进一步帮助比较不同实验组的 DNA 损伤。与传统的 DNA 损伤检测方法相比, 彗星试验具有直接、灵敏、价廉、相对简单等特点。
放疗和化疗是癌症治疗的共同策略, 在染色体上生成单链和双链 DNA 断裂6。最近的进展, DNA 修复抑制剂允许更有效的毒性效果的联合化疗, 因此, 可能会减少系统性副作用, 如贫血, 感染和骨髓抑制7,8. 在这项研究中, 我们展示了一个聚 (ADP-核糖) 聚合酶 (PARP) 抑制剂, olaparib (法律厅)9的研究。PARP 是一种丰富的核蛋白, 通过形成聚 (ADP-核糖) 聚合物10, 负责 DNA 基础切除修复。莫是一种口服的烷剂, 广泛应用于胶质瘤患者的治疗。通过使用彗星试验来量化 dna 损伤, 我们证明了 olaparib 与莫的结合, 深刻地增强了胶质瘤细胞的 dna 损伤, 这表明 olaparib/莫联合疗法是治疗胶质瘤的有效策略,与单独的莫11相比。

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evaluating in vitro dna damage using comet assay

dna 损伤是每个细胞在其生命周期中常见的现象, 它被定义为基因组 dna 化学结构的改变。癌症治疗, 如无线电和化疗, 引入了大量额外的 DNA 损伤, 导致细胞周期阻滞和细胞凋亡, 以限制癌症的进展。在实验性癌症治疗过程中, DNA 损伤的定量评估是证明毒性剂有效性的关键步骤。在这项研究中, 我们的重点是单细胞电泳分析, 也称为彗星检测, 它可以量化单和双 DNA 断裂体外。彗星试验是一种高效、易于执行的 DNA 损伤定量方法, 具有较低的时间/预算要求和较高的重现性。在这里, 我们通过评价 olaparib/莫联合疗法对 U251 胶质瘤细胞的毒性作用, 强调了彗星试验在临床前研究中的效用。

本协议描述了彗星试验执行和数据分析的 step-by 步骤工作流 (图 1)。从碱性和中性彗星的结果表明, 阿霉素治疗的 U251 细胞 (1 µM, 20 h) 的彗星尾巴较长, 并有较高的 dna 强度, 表明由于化疗的碎片 dna 大量积累 (图 2)。
对碱性或中性彗星的定量分析表明, 在阿霉素治疗后彗星尾形成明显?...

Watch this Scientific Journal Video about Evaluating In Vitro DNA Damage Using Comet Assay at JoVE.com

Evaluating In Vitro DNA Damage Using Comet Assay

利用彗星试验评价体外DNA 损伤

彗星试验是一种有效的方法来检测 dna 损伤, 包括单和双 dna 断裂。我们描述了碱性和中性彗星检测肿瘤细胞 DNA 损伤, 以评估化疗的治疗效果。

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Evaluating In Vitro DNA Damage Using Comet Assay

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This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

人类结肠癌细胞DNA损伤与修复福西的免疫荧光成像

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

使用胶原基支架的神经母细胞瘤三维 体外 仿生模型

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

使用患者源性肿瘤类器官的高通量 体外 检测

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...