| Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing
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Author:
Date:
2017-06-05
[Abstract] The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical and NGS-based methods, DNA combing provides unique information on cell-to-cell variations in DNA replication profiles, including initiation and elongation. Finally, this assay can be used to monitor the effect of DNA lesions on fork progression, arrest and restart.
[摘要] DNA梳理方法允许在沿着硅烷涂覆的玻璃盖玻片拉伸的单个DNA分子的水平上分析DNA复制。在DNA提取前，进行的DNA合成用胸苷的卤化类似物标记。使用特异性抗体通过免疫荧光可视化复制轨迹。与生物化学和基于NGS的方法不同，DNA梳理提供了DNA复制谱中细胞间细胞变化的独特信息，包括引发和延长。最后，该测定可用于监测DNA损伤对叉进展，停止和重新启动的影响。
背景 在称为复制起点的真核染色体上的数千个位点处启动DNA合成。原始激活遵循由检查点激酶和染色质的表观遗传修饰（Prioleau和MacAlpine，2016）控制的定义良好的复制计时程序。复制叉在正常S阶段经常停顿。叉停止是由多个事件引起的，例如DNA损伤，紧密结合的蛋白质复合物和高表达基因的转录（Tourriere和Pasero 2007; Zeman and Cimprich，2013）。真核生物已经制定了不同的策略来应对这种复制压力，包括修复机制来重新启动捕获的叉子和激活休眠复制起源以抢救终末抓捕的叉。
＆NBSP; ...
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| In vivo Neurogenesis
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Author:
Date:
2013-08-05
[Abstract] This protocol shows how to characterize the dynamics of hippocampal neurogenesis in the adult mouse by describing preparation of brain tissue, immunofluorescence of brain sections and confocal stereotactic cell counting.
[摘要] 该协议显示如何描述成年小鼠海马神经发生的动力学描述大脑组织的准备，脑切片的免疫荧光和共聚焦立体定向细胞计数。
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| Cell Proliferation Assay by Flow Cytometry (BrdU and PI Staining)
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Author:
Date:
2012-04-05
[Abstract] Cell Proliferation assays include an important set of fluorescence-based tests that can monitor cell health and cell division by evaluating DNA synthesis through thymidine incorporation. Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) is a synthetic nucleoside that is an analogue of thymidine. BrdU is commonly used in the detection of proliferating cells in living tissues. BrdU can be incorporated into the newly synthesized DNA of replicating cells (during the S phase of the cell cycle), substituting for thymidine during DNA replication. Antibodies specific for BrdU can then be used to detect the incorporated chemical, thus indicating cells that were actively replicating their DNA. Binding of the antibody requires denaturation of the DNA, usually by exposing the cells to acid or heat. ...
[摘要] 细胞增殖测定包括一组重要的基于荧光的测试，可通过胸苷掺入评估DNA合成来监测细胞健康和细胞分裂。 溴脱氧尿苷（5-溴-2'-脱氧尿苷，BrdU）是是胸苷类似物的合成核苷。 BrdU通常用于检测活组织中的增殖细胞。 BrdU可以掺入到复制细胞的新合成的DNA中（在细胞周期的S期期间），在DNA复制期间替代胸苷。 然后可以使用BrdU特异性的抗体来检测掺入的化学物质，从而指示活跃地复制其DNA的细胞。 抗体的结合需要DNA变性，通常通过将细胞暴露于酸或热。 通常通过用缀合物抗BrdU抗体和DNA染料碘化丙啶（PI）标记以进行细胞周期分析，在流式细胞术中分析BrdU的掺入。
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