彗星试验测定DNA损伤的海马神经元
1Department of Radiation Oncology,University of Alabama-Birmingham, 2Department of Radiation Oncology,The Ohio State University Medical School, 3Department of Cell Biology, and Pharmacology and Toxicology, Comprehensive Cancer Center,University of Alabama at Birmingham School of Medicine, University of Alabama-Birmingham
Summary
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。
Abstract
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。
Protocol
1。细胞培养的文化神经细胞和治疗自己的需要。 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。 附带在1,000 xg离心5分钟。 吸介质。 在PBS中重悬细胞。 附带在1,000 xg离心5分钟。 吸媒体和新鲜的PBS重悬细胞。 使用血球计数器或您首选的细胞?…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name of the reagent | Company | Catalog number | Comments (optional) |
| 1.5 ml tubes | Santa Cruz Biotechnology, Inc | Sc-200271 | |
| 10X TBE (Tris base, boric acid, EDTA) | Fisher Scientific | BP13331 | |
| 15 ml tube | Fisher Scientific | 0553851 | |
| Agarose | Sigma | A5093 | |
| Aluminum foil | Fisher Scientific | 01213101 | |
| Beaker | Fisher Scientific | FB102300 | |
| Centrifuge | Thermo Scientific | 75004261PR | |
| Comet Assay software | Comet Assay IV Image Analysis System | ||
| Cylinder | Fisher Scientific | 08555F | |
| EDTA | GIBCO | 15575 | |
| Electrophoresis chamber | Thermo Scientific | 09528101 | |
| Ethanol | Fisher Scientific | A407P4 | |
| Fluorescent microscope | Zeiss Axio Vision | Any fluorescent microscope with green filter will suffice | |
| Hemacytometer | Fisher Scientific | 0267152 | |
| Low melting point agarose | Promega | V2111 | |
| Microwave | Sears | ||
| Phosphate buffered saline, calcium free, magnesium free | HyClone | SH3025601 | |
| Power supply | BioRad | 1645050 | |
| Refrigerator | Sears | ||
| Ruler | Staples | ||
| Slide | Fisher Scientific | 12550143 | |
| Sodium chloride | Sigma | S7653 | |
| Sodium hydroxide | Sigma | S5881 | |
| Sodium lauryl sarcosinate | Fisher Scientific | S529 | |
| Sybr green | Invitrogen | S7585 | |
| Tray | Fisher Scientific | 15242B | |
| Tris Base | Sigma | T6066 | |
| Triton-X 100 | Sigma | T8787 | |
| Trypsin | HyClone | SH3023601 | |
| Vortex | Fisher Scientific | 02216108 | |
| Water bath | Fisher Scientific | 154622Q |
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Cite This Article
Nowsheen, S., Xia, F., Yang, E. S. Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay. J. Vis. Exp. (70), e50049, doi:10.3791/50049 (2012).