我们感谢乔尔 Sanneman 和 Philine 博士 Wangemann 的共焦显微镜核心, 由堪萨斯州立大学兽医学院资助, 他们支持的努力开发这种技术。pCBASceI 是玛丽亚 Jasin (Addgene 质粒号 26477) 30 的礼物。U2OS DR-GFP 细胞是玛丽亚 Jasin18的一种礼物。

请注意, 本协议是为包含 SceI 识别站点18的 U2OS 单元格编写的。细胞不需要 U2OS, 但必须包含 SceI 的网站。根据所使用的细胞类型, 可能需要对该协议进行调整 (例如, 种子细胞数和潜伏期)。
1. 定义争端修复复杂地层的动力学
<ol><li>在10厘米的组织培养板上生长 U20S-DRGFP 细胞, 直到85–90% 汇合。</li>
	<li>去除培养基, 在室温下 (RT) 在3毫升 EDTA 中孵育2分钟。</li>
	<li>用1毫升的胰蛋白酶取代 EDTA, 在37摄氏度孵育5分钟. 确保细胞与平板表面分离, 通过显微镜观察。中和胰蛋白酶与5毫升 Dulbecco 的改良鹰培养基 (DMEM) 补充10% 胎牛血清 (血清) 和1% 青霉素/链霉素。</li>
	<li>用 hemocytometer 和台盼蓝排除法确定悬浮液中细胞的浓度。</li>
	<li>种子 6 x 103细胞/井在200µL 的玻璃底部96井板。另外, 种子 4 x 106细胞在8毫升在12毫米盖玻片放置在 10 cm 板材。 注意: 每个井或盖玻片表示单个时间点, 包括控件。</li>
	<li>在37摄氏度和 5% CO2的夜间生长细胞。</li>
	<li>诱导 DSBs
	<ol>
<li>将介质从96井 plate/10 cm 盘中更换为 h2O2稀释至浓度为25µM 与 DMEM + 10% 血清和孵育37摄氏度 1 H。</li>
		<li>用 1x PBS 清洗细胞 3x, 并用新鲜的 DMEM 补充10% 血清和1% 青霉素/链霉素。孵化细胞在37°c 为 0, 1, 4, 8, 24, 和 48 h. 确保每项试验都使用 h2O2的新鲜稀释。 注意: 为了避免在这里使用的 H2O2浓度的细胞中杀死癌细胞, 要确保损伤的剂量足够低, 这样大的细胞就能避免细胞凋亡和衰老。观察恢复过程是至关重要的。这是最好的实现通过测量灵敏度的范围内的剂量。进行 MTT 试验或其他细胞活性测定, 以确定可以使用的过氧化氢的最小浓度。</li>
	</ol></li>
	<li>固定和 permeabilizing 细胞
	<ol>
<li>用 1x PBS 冲洗细胞3次。从10厘米的板上取下一个盖玻片, 并将其放在24井板的一个井中固定。使用针和镊子小心地去除盖玻片和避免刮伤细胞从盖玻片表面。</li>
		<li>在 1 h2O2曝光和新鲜 DMEM 替换 (0、1、4、8、24和 48 h) 后的指定时间, 用4% 多聚甲醛 (煤灰) 孵化15分钟的细胞。</li>
		<li>用 PBS 清洗固定盖玻片3次。Permeabilize 在 1x PBS 中稀释了0.5% 非离子洗涤剂的细胞。孵育15分钟在 RT, 前洗涤3x 与 PBS。 注: 通透后, 盖玻片的贮存在 PBS 中可能在4摄氏度, 至少一周。</li>
	</ol></li>
	<li>修复复杂可视化
	<ol>
<li>阻止96井板/盖玻片在溶液中3% 牛血清白蛋白 (bsa) 和0.1% 非离子洗涤剂稀释在 1x PBS (3% BSA 溶液) 为 1 h 在 RT。</li>
		<li>孵化与主要抗体靶的修复蛋白的利益, 稀释根据制造商的规格在 3% BSA 解决方案为1小时的 RT, 前清洗3倍, 1x PBS。</li>
		<li>在 3% BSA 溶液中, 根据制造商的协议, 孵育96井板/盖玻片和二级抗体稀释。确认二次显影没有重叠的激发或发射谱。</li>
		<li>在洗涤细胞与 PBS 3 次添加 DAPI, 根据制造商的规格稀释 1x PBS 和孵化在 RT 5 分钟。</li>
		<li>将盖玻片安装在幻灯片上, 并放置一个安装代理, 确保单元格端面朝下。仔细按盖玻片, 用擦拭剂浸泡任何多余的安装代理。</li>
		<li>用快干透明指甲油密封盖玻片, 并确保盖玻片与滑轨完全密封。</li>
		<li>通过聚焦在 DAPI 通道上的共焦显微镜图像, 在其他通道 (有兴趣的 DNA 修复基因的信号), 以避免引入偏见的实验。</li>
		<li>或者, 图像可以获得通过采取 Z 部分的期望区域和冷凝图像到一个单一的平面, 以可视化所有可检测的病灶。 注: 如果在选定的时间点未观察到争端解决方案或感兴趣的蛋白质, 则在最初选择广泛的时间点 (0–48 h 后的 DSBs) 时确定何时解决问题。兴趣的时间点会因其诱导的方法和兴趣的修复蛋白而异。</li>
	</ol></li>
	<li>使用自由 ImageJ 软件来定义免疫荧光显微图像中的细胞核。 注: 要打开显微镜图像, 必须在 ImageJ 中安装生物格式插件。<ol>
<li>通过将图像文件拖到 ImageJ 窗口中, 或者从 ImageJ 的 "插件" 工具栏中选择 "生物格式导入程序", 打开共焦图像 (. czi 或. tif)。 注意: 将出现 "生物格式导入选项" 窗口。</li>
		<li>选择 "分割通道", 将图像拆分为 "生物格式导入选项" 窗口中的本构 DAPI 和荧光图像。</li>
		<li>从下拉菜单中选择 "颜色选项" 中的 "彩色"。选择 "确定" 以将 DAPI 和组蛋白 H2AX (H2AX) 图像作为单独的窗口打开, 然后选择 DAPI 图像。</li>
		<li>选择 "图像" 工具栏和 "调整阈值" 选项以选择原子核。 注意: 将出现一个 "阈值" 窗口。</li>
		<li>通过将 "阈值" 窗口的底部条完全向右滑动来调整图像阈值。调整顶部条形图, 直到原子核以不同的轮廓显示为完全红色, 背景为黑色。请勿选择 "应用"。关闭 "调整阈值" 窗口。</li>
		<li>选择 "分析" 工具栏和 "分析粒子" 选项。请参阅屏幕上出现的 "分析粒子" 窗口。</li>
		<li>输入原子核的最小大小。 注: 低于所输入尺寸的粒子将不会算作原子核。</li>
		<li>从 "显示" 下拉菜单中, 选择 "轮廓"。选择 "添加到管理器" 选项。单击 "确定" 打开 "ROI 管理器" 窗口。 注意: 原子核的轮廓将出现在一个单独的窗口中。</li>
		<li>为可以从 "ROI 管理器" 窗口中选择的原子核分配编号。</li>
	</ol></li>
	<li>使用 ImageJ 量化免疫荧光显微图像 H2AX 病灶。
	<ol>
<li>选择 "H2AX" 灶窗口 (染色荧光共轭二次抗体)。然后, 选择 "进程" 工具栏, 选择 "查找最大值" 以找到原子核内的焦点。 注意: "查找最大值" 窗口将打开。</li>
		<li>从 "输出类型" 下拉菜单中选择 "单点" 选项, 然后选择 "预览点选择" 以可视化最大值/焦点。根据图像的荧光强度输入 "噪声耐受" 值 (图 1)。检查带有小黑点的白色窗口的外观。 注意: 这些点表示已检测到的 H2AX 焦点/极大值。对于正在分析的所有图像, 必须保持常量的噪声容差值。低/高噪声耐受将描绘在原子核内的太多/很少的极大值 (病灶), 并不会准确地表示核内的病灶。</li>
		<li>从 "ROI 管理器" 窗口中选择 H2AX 焦点必须量化的原子核。通过检查 "全部显示" 选项选择所有原子核。</li>
		<li>选择 "度量值" 以测量选定原子核中最大值/焦点的个数。 注意: 最大值/焦点的数目显示为255的倍数,即, 一个最高的 "RawIntDen" (集成密度) 读数为255。</li>
		<li>复制 "RawIntDen" 值并将其粘贴到软件中进行数据分析。</li>
	</ol></li>
</ol>2. 对长期双链 DNA 断裂的定位分析
<ol><li>96-井板/盖玻片上的种子细胞, 如1.1 和1.2 节所述。</li>
	<li>
双链 DNA 断裂诱导的研究
	<ol>
<li>染细胞与 I-SceI 表达载体使用脂质为基础的转染试剂根据制造商的规格。在转染后等待 24–72, 以最大化限制性表达式。确定转染是否成功, 通过定位一个奇异的大核γ H2AX 焦点细胞。</li>
	</ol>
</li>
	<li>
获取图像
	<ol>
<li>修复, permeabilize, 块, 并清洗96井板/盖玻片, 如第1.8 节所述。</li>
		<li>联合孵化细胞与抗体抗γ H2AX 和修复蛋白的兴趣 (这里, 使用的主要抗体对 RAD51) 稀释根据制造商的规格, 在 3% BSA 解决方案。</li>
		<li>用 1x PBS 清洗96井板/盖玻片3次。在 3% BSA 溶液中, 根据制造商的协议, 孵育96井板/盖玻片和二级抗体稀释。确认二次显影没有重叠的激发或发射谱。</li>
		<li>识别具有大核γ H2AX 焦点的细胞。只拍这些细胞的照片, 以避免偏见。</li>
	</ol>
</li>
	<li>
长效 I. SceI 诱发病灶的分析
	<ol>
<li>通过计算有大γ H2AX 灶的细胞数量和感兴趣的修复蛋白的共同局部病灶, 手动分析持久灶。</li>
	</ol>
</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATM+Phosphorylates+Histone+H2AX+in+Response+to+DNA+Double-strand+Breaks.">ATM Phosphorylates Histone H2AX in Response to DNA Double-strand Breaks.</a> J Biol Chem. 276 (45), 42462-42467 (2001).</li><li>Britton, S., Coates, J., Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+high-resolution+imaging+of+Ku+foci+to+decipher+mechanisms+of+DNA+double-strand+break+repair.">A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair.</a> J Cell Biol. 202 (3), 579-595 (2013).</li><li>Landthaler, M., Shen, B. W., Stoddard, B. L., Shub, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=I-BasI+and+I-HmuI:+two+phage+intron-encoded+endonucleases+with+homologous+DNA+recognition+sequences+but+distinct+DNA+specificities.">I-BasI and I-HmuI: two phage intron-encoded endonucleases with homologous DNA recognition sequences but distinct DNA specificities.</a> J Mol Biol. 358 (4), 1137-1151 (2006).</li><li>Lackner, D. H., 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+generic+strategy+for+CRISPR-Cas9-mediated+gene+tagging.">A generic strategy for CRISPR-Cas9-mediated gene tagging.</a> Nat Commun. 6, 10237 (2015).</li><li>Chan, S. H., Stoddard, B. L., Xu, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+and+engineered+nicking+endonucleases--from+cleavage+mechanism+to+engineering+of+strand-specificity.">Natural and engineered nicking endonucleases--from cleavage mechanism to engineering of strand-specificity.</a> Nucleic Acids Res. 39 (1), 1-18 (2011).</li></ol>

作者没有什么可透露的。

dna 损伤修复的一般分析和双链 DNA 断裂的修复具体来说是一个活跃的研究领域, 因为它的后果跨越肿瘤发生到基本生物学6,20。本手稿详述了一种方法, 准确地剖析了 RAD51 和γ H2AX 蛋白对 DSBs 通过 HR 的分辨率的贡献. 展望未来, 此方法可用于阐明修复蛋白在 DSBs14中的附加功能。修复动力学的比较和对 SceI 诱导的 DSBs 与细胞之间的控制细胞...

每天, 人体内的每一个细胞都被轰炸, 估计有1万的 DNA 病变1。这种存在的威胁使我们面临突变、肿瘤以及细胞死亡的风险。为了保护基因组的保真度, 哺乳动物细胞进化成了一系列复杂的蛋白质联想和修饰对 DNA 损伤的反应。这种反应被组织成多个途径, 统称为 DNA 损伤反应 (DDR)2,3。复员方案包括 dna 修复蛋白在 dna 损伤的积累, 在世俗和空间上协调。DDR 经常诱发细胞周期骤停, 以避免在复制受损 DNA2、4、5时可能发生的损伤传播或强化。反过来, 细胞活力也必须在修复完成后关闭解除修复复合体的细胞周期停止。
在各种类型的 DNA 损伤中, DSBs 是最有害的。未能修复 DSBs 可能导致染色体重排或大规模删除, 如失去整个染色体武器。DSBs 的修复分为两个路径6,7,8。同源重组 (HR) 要求姐妹染色体作为 DNA 模板使用, 因而被限制在细胞周期9,10的晚 S 和 G2/M 阶段。非同源端连接 (NHEJ) 没有这些限制, 但在修复 DSBs11、12时可能会导致小的删除。
特别报告人修复和复员方案一般是积极的调查领域。尽管被组织成很方便的分离通路, 却有大量的冗余。事实上, 许多蛋白质 (例如 BRCA1、BRCA2 和 RPA 复合体) 都涉及多个路径13、14、15、16。一条通路修复病变, 可导致损伤中间体, 必须由另一通路14修复。这些途径交织在一起, 结合他们的复杂任务, 将正确的蛋白质在正确的地方适当地为必要的时间, 需要一个多层次的管理过程。
最近的一份报告强调了复员方案错综复杂的情况, 表明修复复杂的形成、解决和定位可以分别被削弱17。下面的协议的总目标是彻底解剖细胞修复争端的能力。利用免疫荧光显微镜, 在 DSBs 诱导后的代表性时间点可以可视化损伤部位的修复蛋白积累。
这种技术对常用的方法有几个优点。通常, 在单个时间点进行修复, 不能代表修复配合物的装配和离解的动态过程。从初始激活到完全分辨率, 观察整个修复范围, 可确保延迟修复不认错为完全抑制。相反, 它保证了无法停用的修复响应的诱导, 不认错为正常或过度激活。
然而, 延迟蛋白复合物的形成和修复蛋白的错误定位可能与这种方法没有明确的区别。为了确定修复蛋白在定位时是否与延迟有关, 可以通过酶解细胞 DNA 来引入 "长效" 的分裂分子。所产生的病变是裁每次修复时, 导致一个明显的大核修复重点和消除时间限制, 从招聘。这可以通过修改现有的方法, 使用罕见的切割限制性 (如SceI) 来诱导一个长期的争端解决办法来实现。DSBs 的寿命使免疫荧光显微镜能直观地显示出难以捉摸的修复蛋白。增强的丰度还可以改善可视化, 当检测受到限制的抗体质量, 这一特点, 可能是有用的, 当较小的研究蛋白质被认为对 DNA 修复的影响。
值得注意的是, 我们提供了一个自由图像处理和分析软件 (如ImageJ) 的明确指令。这就消除了图像分析中的一个主要的金融障碍, 打开了对更广泛的受众进行 DNA 损伤修复的审问。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12mm Coverslips</td>
			<td>VWR</td>
			<td>89015-725</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde (PFA)</td>
			<td>ThermoFisher Scientific</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>VWR</td>
			<td>82050-892</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well glass bottom plate</td>
			<td>Cellvis</td>
			<td>P96-1.5H-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-H2AX Alexa488</td>
			<td>EMD Millipore</td>
			<td>05-636-AF488</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Rad51 (D4B10)&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>8875S</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-formats plugin for ImageJ</td>
			<td>National Institute of Health (NIH)&#160;</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/plugins/index.html</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>VWR</td>
			<td>97061-416</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, High Glucose</td>
			<td>ThermoFisher Scientific</td>
			<td>12100046</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15576-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>VWR</td>
			<td>89510-194</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG Alexa594</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;A-11012&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide</td>
			<td>sigma-Aldrich</td>
			<td>216763-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>National Institute of Health (NIH)&#160;</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>I-SceI Expression Vector</td>
			<td>Addgene</td>
			<td>26477</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail Polish- Insta Dri</td>
			<td>Sally and Hansen</td>
			<td>Clearly Quick (103)</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Bio Basic</td>
			<td>PD8117</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Reagent</td>
			<td>Life Technologies</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T4049-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TurboFect Transfection Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>R0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing dna repair processes at transient and long-lasting double-strand dna breaks by immunofluorescence microscopy

在 DNA 中修复双链断裂 (DSBs) 是一个高度协调的过程, 它有必要形成和解决多蛋白修复复合体。这一过程是由无数的蛋白质, 促进蛋白质的联想和解除这些病变。在很大程度上, 由于能够执行一个巨大的蛋白质库功能屏幕, 有一个更大的赞赏的基因, 双链 DNA 断裂修复。经常敲除或化学抑制剂屏幕识别参与修复过程中的蛋白质, 使用增加的毒性作为一个标记的蛋白质, 这是需要的争端处理修复。功能分析虽然对于确定涉及维持基因组保真度的新的细胞蛋白很有用, 但需要确定感兴趣的蛋白质是否促进修复复合体的定位、形成或解决。
通过免疫荧光显微镜可以很容易地检测到修复蛋白的积累。因此, 这些蛋白在 dna 损伤部位的联想和解除可以通过在诱导双链 DNA 断裂后观察这些核病灶来获得。这种方法还可以识别错误本地化的修复因子蛋白, 如果修复缺陷不同时发生, 修复不完全延迟。在这种情况下, 长时间的双链 DNA 断裂可以通过表达一个罕见的切割限制性 (例如, I-SceI) 在细胞中, 该酶的识别站点已经集成到细胞基因组中来设计。由于忠实的修复将重新引入酶的识别部位, 引发另一轮的分裂, 因此导致的病变特别难以解决。因此, 消除了修复动力学的差异。如果未形成修复配合物, 则会阻碍局部化。本协议描述了确定修复动力学的变化以及修复蛋白质定位所需的方法。

图 1描述了使用 ImageJ 对最大值/焦点量化的正确噪声判别的选择。DAPI 的合并图像和感兴趣的修复蛋白在左面板上。图 1A显示了90的噪声判别, 并标记了正确的焦点数目。在定量过程中不计算边缘上的原子核 (用粉红色箭头描绘) 和原子核外的病灶 (用黄色箭头描绘)。图 1B描述了低噪音公差50。在原子核外和?...

Watch this Scientific Journal Video about Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy at JoVE.com

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

双链 DNA 断裂的修复是一个动态过程, 不仅需要在断裂时形成修复配合物, 而且要解决病变后的解决方法。在这里, 我们使用免疫荧光显微镜的短暂和长期的双滞留断裂作为一个工具来解剖这个基因组维持机制。

基于免疫荧光显微镜的瞬时和持久双链 dna 损伤 dna 修复过程的表征

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

characterizing-dna-repair-processes-at-transient-long-lasting-double

Research

JoVE Journal

Cancer Research

9.0K 次观看.  Kansas State University. 双链 DNA 断裂的修复是一个动态过程, 不仅需要在断裂时形成修复配合物, 而且要解决病变后的解决方法。在这里, 我们使用免疫荧光显微镜的短暂和长期的双滞留断裂作为一个工具来解剖这个基因组维持机制。

视频: Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

Cell-Free DNA Integrity Analysis in Urine Samples

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of cancer, few studies have focused on the potential use of urine cell-free (UCF) DNA. Starting from the hypotheses that normal apoptotic cells produce highly fragmented DNA and that cancer cells release longer DNA, the potential role of UCF DNA integrity was evaluated as an early diagnostic marker capable of distinguishing between patients with prostate or bladder cancer and healthy individuals.
A UCF DNA integrity analysis is proposed on the basis of four quantitative real-time PCRs of four sequences longer than 250 bp: c-MYC, BCAS1, HER2, and AR. Sequences that frequently have an increased DNA copy number in bladder and prostate cancers were chosen for the analysis, but the method is flexible, and these genes could be substituted with other genes of interest. The potential utility of UCF DNA as a source of biomarkers has already been demonstrated for urologic malignancies, thus paving the way for further studies on UCF DNA characterization. The UCF DNA integrity test has the advantage of being non-invasive, rapid, and easy to perform, with only a few milliliters of urine needed to carry out the analysis.

A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

无细胞DNA完整性分析尿样中

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of ...

科研

癌症研究

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

激光微辐照在哺乳动物细胞单、双链断裂修复中的应用

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

Evaluating In Vitro DNA Damage Using Comet Assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Evaluating In Vitro DNA Damage Using Comet Assay

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

利用彗星试验评价体外DNA 损伤

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of &#947;H2AX and 53BP1, the combined analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on &#947;H2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the &#947;H2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of &#947;H2AX and 53BP1.

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of &#947;H2AX and 53BP1.

γH2AX 和53BP1 的免疫荧光显微镜分析 DNA 双链断裂的形成和修复

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

用医用金属丝捕获循环肿瘤细胞的肿瘤细胞: 基于免疫荧光和 DNA 的3D 方法

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a reliable probability of the presence of pre-neoplastic or neoplastic colorectal lesions. This method, called fluorescence long DNA (FL-DNA), is a fast, non-invasive procedure that is an improvement upon the primary prevention system. This method is based on evaluation of fecal DNA integrity by quantitative amplification of specific targets of genomic DNA. In particular, the evaluation of DNA fragments longer than 200 bp allows for detection of patients with colorectal lesions with very high specificity. However, this system and all currently available stool DNA tests present some general issues that need to be addressed (e.g., the frequency at which tests should be carried out and optimal number of stool samples collected at each timepoint for each individual). However, the main advantage of FL-DNA is the possibility to use it in association with a test currently used in the CRC screening program, known as the immunochemical-based fecal occult blood test (iFOBT). Indeed, both tests can be performed on the same sample, reducing costs and achieving a better prediction of the eventual presence of colorectal lesions.

The presented diagnostic FL-DNA kit is a time-saving and user-friendly method to determine the reliable probability of the presence of colorectal cancer lesions.

通过大刀DNA完整性检测评估结肠直肠癌风险和患病率

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

在Vivo免疫荧光定位中用于评估癌症研究中的治疗和诊断抗体生物分布

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is a useful measure to provide an accurate analysis of the methylation status of gastrointestinal (GI) malignancies. Given the multiple potential translational uses of DNA methylation analysis, practicing clinicians and others new to DNA methylation studies need to be able to understand step by step how these genome-wide analyses are performed. The goal of this protocol is to provide a detailed description of how this method is used for the biomarker identification in GI malignancies. Importantly, we describe three critical steps that are needed to obtain accurate results during genome-wide analysis. Clearly and concisely written, these three methods are often lacking and not noticeable to those new to epigenetic studies. We used 48 samples of a GI malignancy (gastric cancer) to highlight practically how genome-wide DNA methylation analysis can be performed for GI malignancies.

Herein, we describe a procedure for genome-wide analysis of DNA methylation in gastrointestinal cancers. The procedure is of relevance to studies that investigate relationships between methylation patterns of genes and factors contributing to carcinogenesis in gastrointestinal cancers.

胃肠癌DNA甲基化基因组全谱分析

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is ...

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

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

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 ...