作者要感谢博士。 Philina S. Lee和艾伦D.格罗斯曼初步培训阿盟在荧光显微镜。作者还感谢博士。梅拉妮Berkmen和肇小林成像的帮助和提示。这项工作是从文学，科学和艺术学院，并在密歇根大学的分子，细胞，和发育生物学部的启动资金支持。

显微镜细胞成长的文化 1。前一两天，影像，准备B.枯草芽孢杆菌菌株，包含你想可视化的翻译融合蛋白。步骤1a和1b的审判轮将是必要的，以确定哪些生长条件提供最好的图像你的应变。在大多数情况下，细胞在指数生长期应成像。 <ol class="ualpha"><li>对于一些B。枯草芽孢杆菌菌株（尤其是株生长不良，由于之间的兴趣和在其内源性的轨迹的绿色荧光蛋白基因的翻译融合整合），成像前两天的初始增长是必要的高质量的图像。在第一天，连胜的B.枯草芽孢杆菌菌株在LB琼脂成像与选择性抗生素（S）在30 ° C过夜孵育第二天，单菌落接种100μL0.85％生理盐水和执行三个10倍连续稀释的LB琼脂平板（选择性抗生素），电镀100μL每个稀释度，其次孵育过夜30 ° C。第三天，选择与光融合细胞生长的稀释板。光一个板块的汇合增长将提供与健康，成倍增长的起动接种潜在收益出色的成像效果（见第4步）的培养基中。这一步是最高质量的FM4 - 64染色膜的成像特别有用。 </li><li>其他B。枯草芽孢杆菌菌株可能只是选择性的LB琼脂培养基单菌落用无菌棒的前一天，影像，并培育在30 ° C过夜翌日，这个板块的细胞，将足以成像接种使用（见第4步）。 注： 为 B 枯草芽孢杆菌在液体培养基中过夜培养，由于不恰当的组合的孢子形成和法定人数的反应，这将导致一个较长时间的滞后期增长 1。 </li></ol> 2。在成像，放置10毫升的S7 50 1-3成125毫升的无菌锥形瓶中中等上午。 3。悬浮细胞S7 50中等至2毫升的LB琼脂平板上单菌落或光的融合细胞获得接种。 4。添加足够的S7 50中细胞接种到含有10毫升S7 50培养基中，在600 nm处测初始光密度 （OD 600）〜0.08至0.1的锥形瓶中。在这一步是很重要，至少有一个中等比例的10倍的空气。因此，我们使用10至12.5毫升接种于125 mL锥形瓶中的培养基。 5。每一种文化，文化℃直至达到一个外径600〜0.5-0.8增长30 。从150-200转每分钟振动水浴是首选。文化与DNA损伤或不匹配诱导剂的挑战，应在早期的指数生长期外径600细胞与治疗所需的额外增长期将达到目标的外径600（见表1） 例如，2 -氨基嘌呤，需要一小时的治疗，因此，2 -氨基嘌呤治疗文化应在外径 600〜0.3-0.4，例如，在外径 600治疗/业绩增长的一小时〜 0.5-0.8， 据此细胞成像。 样品制备 1。吸取300μL培养细胞到1.5 ml离心管中。 2。如果需要细胞膜成像，每个样品中加入1:1000稀释FM4 - 64膜染色（Invitrogen公司），从1毫克/毫升的原液。 FM4 - 64滴定可能需要达到理想的荧光信号。一个典型的滴定范围为1:100，1:1000，并1:10,000。 3。允许样品在室温为10分钟或坐而幻灯片正在准备在1X Spizizens介质，用1％琼脂糖如下所述（见“幻灯片准备”的步骤1）。 4。如果细胞浓度是必需的，我们更倾向于使用一个0.2微米的过滤器与真空仪器的过滤细胞。细胞可以轻轻洗净，从表面过滤器，用PBS，另一种合适的缓冲区或中期之前可视。 幻灯片的制备 1。 1％琼脂糖准备1X Spizizens。 添加5毫升0.5 G琼脂糖股票10X Spizizens至45毫升ddH2O，微波融化。热熔融琼脂糖难以吸管，和凝固的琼脂糖不会进入枪头。琼脂糖应该是平衡的水浴温度〜55-65℃，进入枪头没有在固化<html>一角。 2。绘制1％琼脂糖融化到20μLSpizizens枪头。移液器一个单一琼脂糖溶液下降到每张幻灯片（我们使用15以及幻灯片，见表2）形成较浅的琼脂糖垫（大约1-2μL）。琼脂糖将立即巩固。幻灯片也必须立即作出之前，应用细胞。如果琼脂糖垫仅在室温下离开，他们可以在几分钟之内脱水，变得无法使用。 琼脂糖垫应为本，填写每个很好，但有最低的高度，以避免破坏盖玻片;如果水滴过高（泡沫状），或者如果样品以及边界以外的利差，只需轻扫一个Kimwipe以及清洁和应用新琼脂糖垫。 3。整个300μL样本（见第6步），分布在每个琼脂糖垫到幻灯片。这种细胞的数量应足以支付每口井在幻灯片上。 4。允许加载与细胞坐在室温（23℃）约10分钟的幻灯片。这一步将允许细胞琼脂糖垫成为不动，并准备好用于成像解决。对于特殊需求，这可能需要改变温度，例如，进行实验时，需要一个温度变化 4 。 5。取出垫本身没有吸走多余的介质，从每口井。 6。应用盖玻片的幻灯片，稳固地按压均匀，但幻灯片区域，轻轻划过。检查，以确保盖玻片对幻灯片和盖玻片，是不是上面的幻灯片提出。 细胞的可视化 1。许多不同的荧光显微镜，将工作做好。有100X油浸镜头，这是关键。西蒙斯实验室使用： <ul><li>奥林巴斯BX61显微镜配备1,45 NA 100X TIRFM油浸物镜</li><li>滨松ORCAR 2 CCD相机冷却</li><li> 200流明弧金属（前）光源。 </li><li> GFP（FITC），过滤器激发460-500和510-560排放检测</li><li> FM4 - 64（TRITC），过滤器励磁510-560和572-648排放检测。 </li><li>图像捕获SlideBook 4.2（图1） </li></ul> 2。使用白光的重点带入细胞。 3。每个荧光使用适当的过滤器捕捉的图像。 代表性的成果代表图像显示（图1）。 GFP的疫源地，应明确界定，而应FM4 - 64染色鲜艳和清晰的4-7的。绿色荧光蛋白或膜的图像可以与任何颜色的伪彩色，允许提供最高质量的图像不会丢失任何数据提交。 <img src="/files/ftp_upload/1736/1736fig1.jpg" alt="图1" /> 图1：B 的代表的GFP图像枯草翻译融合蛋白 GFP的蛋白质以绿色显示，而以红色显示FM4 - 64膜染色。白色比例尺表示3微米（一）MutS - GFP [相关基因型：mutS - 绿色荧光蛋白（SPC），amyE：Pspac mutL（CAT）]，在600微克/毫升2 -氨基嘌呤和1毫米的IPTG存在。 （三）FM4 - 64的信号是不是为了更清楚地显示MutS - GFP的灶（二 ）MutL - GFP [相关基因型：mutL：mutL - GFP（SPC）]在2 -氨基嘌呤存在。 DnaX - GFP [相关基因型：dnaX：dnaX绿色荧光蛋白（SPC）]。 （四）RecA蛋白- GFP [相关基因型：recA基因：recA基因，绿色荧光蛋白（SPC）]存在20 ng / mL的丝裂霉素C（E）TAGC - GFP [相关基因型：TAGC：TAGC - GFP（SPC）在1μg/ mL的丝裂霉素C <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td>治疗/浓度或数量</td><td>功能</td><td>孵化时间</td></tr><tr><td>丝裂霉素C（MMC）[20〜150毫微克/毫升] </td><td> DNA的烷基化剂</td><td> 1小时</td></tr><tr><td> 2 - 氨基嘌呤（2 - AP）[600微克/毫升] </td><td>不匹配的诱导</td><td> 1小时</td></tr><tr><td>羟基脲（HU） </td><td>耗尽dNTPs </td><td> 3个小时</td></tr><tr><td>紫外线（UV）20 J/m2 </td><td>胸腺嘧啶胸腺嘧啶二聚体和6-4光化</td><td>因取决于源代码，通常为20秒</td></tr><tr><td>灰色（电离辐射）5至100戈瑞</td><td>双链断裂，单链断裂和基础伤害网站</td><td>因来源不同而异</td></tr></tbody></table>“&gt; 表1总结的DNA损坏和不匹配的诱导治疗 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> Multitest的幻灯片，15 </td><td> MP生物医学</td><td> 6041505E </td><td></td></tr><tr><td>显微镜盖玻片</td><td>费舍尔</td><td> 12 - 544 - B </td><td></td></tr><tr><td>琼脂糖</td><td>费舍尔</td><td> BP160 - 500 </td><td></td></tr><tr><td>丝裂霉素C </td><td>西格玛</td><td> M0503 - 2MG </td><td>诱变剂，戴手套</td></tr><tr><td> 2 - 氨基嘌呤</td><td>西格玛</td><td> A3509 - 250毫克</td><td>手套</td></tr><tr><td>羟基脲</td><td>西格玛</td><td> H8627 - 25G </td><td>手套</td></tr><tr><td> FM4 - 64 </td><td> Invitrogen公司</td><td> T13320 </td><td>对光敏感</td></tr></tbody></table> 表2。特定的试剂和仪器的清单：

<ol>
	<li>Hardwood, C. R., Cutting, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Biological Methods for Bacillus. , (1990).</li><li>Berkmen, M. B., Grossman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+organization+of+the+Bacillus+subtilis+replication+cycle.">Spatial and temporal organization of the Bacillus subtilis replication cycle.</a> Mol. Microbiol. 62, 57-71 (2006).</li><li>Simmons, L. A., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clp+and+Lon+proteases+occupy+distinct+subcellular+positions+in+Bacillus+subtilis.">Clp and Lon proteases occupy distinct subcellular positions in Bacillus subtilis.</a> J. Bacteriol. 190, 6758-6768 (2008).</li><li>Simmons, L. A., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+is+required+for+the+RecA+localization+response+to+DNA+damage+in+Bacillus+subtilis.">Replication is required for the RecA localization response to DNA damage in Bacillus subtilis.</a> Proc. Natl. Acad. Sci. U S A. 104, 1360-1365 (2007).</li><li>Simmons, L. A., Davies, B. W., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=b+Clamp+Directs+Localization+of+Mismatch+Repair+in+Bacillus+subtilis.">b Clamp Directs Localization of Mismatch Repair in Bacillus subtilis.</a> Mol. Cell. 29, 291-301 (2008).</li><li>Simmons, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+responses+to+double-strand+breaks+between+Escherichia+coli+and+Bacillus+subtilis+reveals+different+requirements+for+SOS+induction.">Comparison of responses to double-strand breaks between Escherichia coli and Bacillus subtilis reveals different requirements for SOS induction.</a> J. Bacteriol. 191, 1152-1161 (2009).</li><li>Smith, B. T., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+mismatch+repair+in+bacterial+cells.">Visualization of mismatch repair in bacterial cells.</a> Mol. Cell. 8, 1197-1206 (2001).</li></ol>

试验和错误，需要找到每一株最高质量的图像的曝光条件，我们发现，1毫秒是适当的白色光图像，而100到2000毫秒的曝光是适当绿色荧光蛋白（FITC）和FM4 - 64（TRITC ）图像。取决于所使用的成像设备，曝光时间会有所不同。我们建议每15井显微镜幻灯片应变使用最简单的成像的，垫垫边境细胞的质量和扩散可能株分化的复杂性，多株，如果是同一张幻灯片上。图像质量直接取决于琼脂糖垫细菌休息...

具体的解决方案食谱1： 10倍的S7 50盐 0.5中号MOPS 100毫米硫酸铵 50毫米磷酸二氢钾 过滤消毒，包装的S7存储前50媒体在铝箔 100X金属 0.2 M MgCl 2的 70毫米氯化钙2 5毫米MnCl 2 0.1毫米氯化锌 100微克/毫升硫胺素盐酸 2毫米的盐酸 0.5毫米FECL 3 * DH 2 O的最后一卷 * FECL 3应该是最后添加，以防止沉淀。 经过过滤灭菌，包装100X金属箔的解决方案。 S7 50媒体 1X的S7 50盐 1X金属 葡萄糖1％ 0.1％，谷氨酸 40微克/毫升色氨酸 40微克/毫升的苯丙氨酸 蒸馏水H 2 O至终体积 10X Spizizens（克/升） 151.4 mM的硫酸铵（20G / L） 803.8毫米磷酸二氢钾（140克/ L） 440.9 mM磷酸钾二元（60G / L） 34.0 mM柠檬酸钠（10g / L的） 16.6毫米硫酸镁 （2G / L） DH 2 O的最后一卷 过滤消毒

imaging mismatch repair and cellular responses to dna damage in bacillus subtilis

原核生物和真核生物都通过一个复杂的生理变化响应DNA损伤。在基因的表达，对现有蛋白质的再分配，以及新的蛋白质复合物的组装的改变，可以刺激多种DNA损伤和不匹配的DNA碱基对。荧光显微镜已被用来作为一个强大的可视化和量化这些和其他的反应，DNA损伤和监测在一个活细胞的亚细胞结构的复杂中的DNA复制状态的实验工具。荧光记者蛋白质和DNA复制和修复机制的组成部分之间的翻译融合已被用来确定的线索，其同源性病变的目标DNA修复蛋白<em&gt;在体内</em&gt;，并了解如何将这些蛋白质在细菌细胞内举办。此外，与DNA损伤诱导促销员的转录和翻译融合显示已激活细胞内人口的遗传毒性应激反应。在这次审查中，我们提供了一个详细的协议，用荧光显微镜图像的单个细菌细胞的DNA修复和DNA复制复合物的组装。特别是，这个工作重点成像错配修复蛋白，同源重组，DNA复制和一个SOS -诱导蛋白<em&gt;枯草芽孢杆菌</em&gt;。此处描述的步骤都可以适合在各种不同的细菌种类成像蛋白复合物。

Watch this Scientific Journal Video about Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis at JoVE.com

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

一个详细的协议是描述成像的DNA修复复合物的真正形成时间<em&gt;枯草芽孢杆菌</em&gt;细胞。

成像错配修复和DNA损伤的细胞反应枯草芽孢杆菌</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

imaging-mismatch-repair-cellular-responses-to-dna-damage-bacillus

Research

JoVE Journal

Biology

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

10.9K 次观看.  University of Michigan-Ann Arbor. 一个详细的协议是描述成像的DNA修复复合物的真正形成时间枯草芽孢杆菌细胞。

视频: Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

二维和三维活细胞成像的DNA损伤反应蛋白

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

科研

生物学

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

基因组DNA富集转座为基础的技术的整个序列的农工商分析<em&gt; BRCA1基因</em&gt;<em&gt; BRCA2</em&gt;和9个基因参与DNA损伤修复

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

在联合电子能谱成像miniSOG标记的DNA修复蛋白可视化（ESI）

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

测量DNA损伤与修复小鼠脾细胞慢性后<em&gt;在体内</em&gt;暴露于β和γ辐射剂量很低

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

体内碱性彗星试验和酶修饰碱性彗星试验测量DNA链断裂和DNA氧化损伤大鼠肝

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

单细胞微流控芯片分析枯草芽孢杆菌

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

可视化和量化基于内分泌酶的站点特定DNA损伤

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

体外 E3泛素连接酶功能分析

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...