澳大利亚核科学与工程学院的支持，是公认的事实。 TCK的是AINSE奖项的收件人。表观医学实验室是支持由国家卫生和澳大利亚的医学研究理事会（566559）。 LM是由墨尔本研究所（墨尔本大学）和生物医学成像的华润补充奖学金支持。莫纳什显微成像（DRS斯蒂芬科迪ISKA卡迈克尔）的支持是这项工作的宝贵。蒙脱土是奥尔加Sedelnikova博士从美国国立卫生研究院的专家援助和意见表示感谢。

组织<ol><li>肺组织，在模具中嵌入最佳切削温度（OCT）的化合物4583，切片（5μm）的使用CM徕卡1950年的低温恒温器，安装到聚- L -赖氨酸的幻灯片。 </li></ol>冷冻样品在默多克儿童研究所获得安博士劳斯莱斯。肺组织是未经处理的BALB / c小鼠的慢性呼吸道过敏性疾病，显示许多人类哮喘6的病理特征的小鼠模型。默多克儿童研究院坚持实践澳大利亚的关心和用于科研目的的实验动物的使用守则“动物伦理委员会提出的准则和法规的规定进行了动物实验。 <ol start="2"><li>部分被允许在室温空气干燥1小时。 </li></ol>免疫荧光染色<ol><li>固定2％（W ​​/ V）多聚甲醛磷酸盐缓冲液（PBS）20分钟。 </li><li>部分在PBS洗涤，每次15分钟的平衡。 </li><li>部分均采用70％乙醇20分钟，由三个用PBS洗涤，每次10分钟（冷冻至-20 ° C）。 </li></ol>此时的幻灯片可在4 ° C储存在密封的容器内长达2个星期。 <ol start="4"><li>切片，用PBS洗三次，每次10分钟，然后用新鲜PBS中含有8％的PBS中含0.5％Tween - 20与0.1％的牛血清白蛋白准备100μL封锁的Triton X - 100在室温下1小时（PBS - TT） 。 所有孵化加湿染色槽。 </li><li>第5分钟，用PBS - TT分别洗净，和疏水性的外围组织周围标有巴氏笔。 </li><li>通风报信多余的缓冲液和主多克隆抗γH2AX抗体（稀释1％BSA的PBS 1:500;微孔）100μL，在室温下孵育2小时每节。 育才小学抗体可能会在一夜之间被执行4 ° C。 对于进一步的证据，即γH2AX灶代表双链断裂，主承认DSB修复蛋白的抗体也可使用。例如，γH2AX和磷酸化的ATM的合作，本地化是一个常用的组合。 </li><li>节在PBS - TT洗两次，每次5分钟，并与二级抗体100μL（的Alexa Fluor 488山羊抗兔IgG 1:500稀释，在1％BSA; Invitrogen公司）孵育1小时在室温下在黑暗中。 （稀释后的抗体，整个过程被蒙在鼓里）。 </li><li>洗净切片，用PBS - TT三次，每次5分钟，并孵育0.5mg/ml的核糖核酸酶A为30分钟，在37 ° C。 </li><li> Vectashield介质中含有碘化丙啶核counterstaining和安装。 </li><li>用指甲油密封的幻灯片，并储存于4 ° C黑暗环境中分析前通宵。 </li></ol>显微镜/分析<ol><li>蔡司LSM510元共聚焦显微镜是用来获取图像，使用标准的绿色荧光蛋白（γH2AX - 的Alexa Fluor 488山羊抗兔IgG）和PI（543 nm）的激光。 </li></ol>在Z系列模式图像获得一个0.5微米的一步大小。在分析过程中，个别飞机卷积和堆叠，以产生最大的投射影像，以减少灶重叠。 创建一个最大投射影像的前一个粒子计数的方法，只应在使用薄的部分（如5微米）和背景染色是微不足道的情况下使用。凡具有显着的背景较厚部分必须加以分析，如3D对象计数器Bolte和Cordelieres（2006）描述的更复杂的分析，应使用 7 。 <ol start="2"><li> Metamorph分子器件，（美国）是用于创建合并（红色和绿色）图像定量灶的数字。 </li></ol>虽然Metamorph可用于定量灶数目，通常在使用组织病灶手动（眼），用于更精确的计算部分。 定量感兴趣的特定的细胞类型可以选择，例如肺上皮细胞。这可能是基于组织学和苏木和HE染色的连续切片。

<ol>
	<li>Rogakou, E. P., Boon, C., Redon, C., Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Megabase+chromatin+domains+involved+in+DNA+double-strand+breaks+in+vivo.">Megabase chromatin domains involved in DNA double-strand breaks in vivo.</a> J. Cell Biol. 146, 905-916 (1999).</li><li>Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma+H2AX+and+cancer.">Gamma H2AX and cancer.</a> Nature Reviews Cancer. 8 (12), 957-967 (2008).</li><li>Dickey, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2AX:+functional+roles+and+potential+applications.">H2AX: functional roles and potential applications.</a> Chromosoma. 118 (6), 683-692 (2009).</li><li>Bhogal, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+residual+gamma-H2AX+foci+in+murine+skin+are+dose+responsive+and+predict+radiosensitivity+in+vivo.">Late residual gamma-H2AX foci in murine skin are dose responsive and predict radiosensitivity in vivo.</a> Radiat Res. 173 (1), 1-9 (2010).</li><li>Redon, C. E., Dickey, J. S., Bonner, W. M., Sedelnikova, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma-H2AX+as+a+biomarker+of+DNA+damage+induced+by+ionizing+radiation+in+human+peripheral+blood+lymphocytes+and+artificial+skin.">gamma-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skin.</a> Adv Space Res. 43 (8), 1171-1178 (2009).</li><li>Locke, N. R., Royce, S. G., Wainewright, J. S., Samuel, C. S., Tang, M. L., L, M. <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+airway+remodeling+in+acute,+subacute,+and+chronic+models+of+allergic+airways+disease.">Comparison of airway remodeling in acute, subacute, and chronic models of allergic airways disease.</a> Am J Respir Cell Mol Biol. 36 (5), 625-632 (2007).</li><li>Bolte, S. &. a. m. p. ;. a. m. p., Cordelieres, F. 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+guided+tour+into+subcellular+colocalization+analysis+in+light+microscopy.">A guided tour into subcellular colocalization analysis in light microscopy.</a> J Microsc. 224 (Pt 3), 213-232 (2006).</li></ol>

这个演示中，我们使用未经处理的BALB / c小鼠，小鼠肺组织的慢性呼吸道过敏性疾病的小鼠模型的目的。我们定量细胞受损的肺相比，未经处理的部分的平均水平略高，每灶数量的差异。具体来说，定量表示6.5元/单元和9.4灶元/单元（每节20细胞人工计数）灶，在未经处理的组织和慢性呼吸道过敏性疾病组织的分别。然而，例如萘，这是众所周知的诱导小鼠肺癌模型的DSBs的DSB诱导剂的使用，会产生...

<table><tbody><tr><td>Bovine Serum Albumin (BSA)</td><td></td><td>Sigma-Aldrich</td><td>A7906</td><td>BSA (1%) is used to block any non-specific antibody binding. Primary and secondary antibodies are diluted in BSA.</td></tr><tr><td>PBS (without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td></td><td>Invitrogen</td><td>17-517Q</td><td></td></tr><tr><td>Optimal Cutting Temperature (OCT) Compound 4583</td><td></td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>Superfrost Plus</td><td>Polysine slides</td><td>Menzel-Glaser</td><td>SF41296SP</td><td>Electrostatically attracts frozen tissue sections and reduces the need for additional coatings and adhesives.</td></tr><tr><td>Tween 20</td><td>Reagent</td><td>Calbiochem</td><td>655205</td><td>Tween 20 (0.5%), is a detergent to permeabilise cells</td></tr><tr><td>Triton X-100</td><td>Reagent</td><td>Sigma-Aldrich</td><td>T8787</td><td>Triton X-100 (0.1%) used to permeabilise cells.</td></tr><tr><td>Paraformaldehyde</td><td>Reagent</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Anti-phospho-&amp;#947;H2AX (rabbit polyclonal antibody)</td><td>Primary Antibody</td><td>Upstate, Millipore</td><td>07-164</td><td>Dilution of primary antibody (1:500), in 1% BSA.</td></tr><tr><td>Alexa Fluor 488 goat anti-rabbit IgG (H+L)</td><td>Secondary Antibody</td><td>Invitrogen</td><td>A-11008</td><td>Dilution of secondary antibody (1:500), in 1% BSA.</td></tr><tr><td>Ethanol (70%)</td><td></td><td>Thermo Fisher Scientific, Inc.</td><td>BSPEL316</td><td></td></tr><tr><td>RNAse A</td><td>Reagent</td><td>Qiagen</td><td>19101</td><td></td></tr><tr><td>Vectashield PI</td><td>Reagent</td><td>Vector Laboratories</td><td>H-1300</td><td>A glycerol based mounting medium containing propidium iodide (a red nuclear stain).This medium must be used with an oil based lense that matches its refractive index.&lt;br/&gt;&lt;br/&gt;Note: DNA stain commonly used: 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Can only be used with microscopes with the appropriate excitation laser.</td></tr><tr><td>Mini PAP Pen</td><td></td><td>Invitrogen</td><td>008877</td><td></td></tr><tr><td>Coverslips (22x50mm)</td><td></td><td>Menzel-Glaser</td><td>CS2250100</td><td></td></tr><tr><td>Coplin Jar, glass</td><td></td><td>Grale Scientific P/L</td><td>1771-OG</td><td></td></tr><tr><td>Staining Trough</td><td></td><td>Grale Scientific P/L</td><td>V1991.99</td><td></td></tr><tr><td>Zeiss LSM 510 Meta Confocal</td><td></td><td>Confocal Microscope</td><td></td><td></td></tr><tr><td>CM Leica 1950 Cryostat</td><td></td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Metamorph</td><td>Software for Imaging analysis</td><td>Molecular Devices</td><td></td><td></td></tr></tbody></table>

quantitation of &gamma;h2ax foci in tissue samples

DNA双链断裂（DSB的），特别是致死和遗传毒性病变，可以产生内源性（生理或病理）的进程或外在因素，特别是电离辐射和radiomimetic化合物。磷酸化的组蛋白H2A的变种，H2AX的丝氨酸139残留，高度保守的C -末端SQEY图案，形成γH2AX，是一种早期反应的DNA双链断裂<sup&gt; 1</sup&gt;。这inosito的磷脂，肌醇3 - 激酶（PI3K）的蛋白质家族，共济失调毛细血管扩张症（ATM）的突变，DNA -蛋白激酶催化亚基和ATM和RAD3相关介导的磷酸化事件（ATR）<sup&gt; 2</sup&gt;。整体而言，在形成离散核γH2AX灶DSB诱导的结果，可以很容易地检测和免疫荧光显微镜定量分析<sup&gt; 2</sup&gt;。由于这个标记独特的特异性和灵敏度，分析γH2AX灶导致广泛的生物医学研究中的应用程序，尤其是在放射生物学和核医学，。 γH2AX灶的定量已在电离辐射诱导的DNA双链断裂的情况下在细胞培养系统最广泛的调查。除了从细胞放射敏感性，免疫检测也被用来评估辐射修改化合物的疗效。此外，γH2AX作为分子标记已用于研究各种DSB诱导化合物的疗效，是最近正预示着衰老和疾病的重要标志，尤其是癌症<sup&gt; 3</sup&gt;。此外，免疫为基础的方法已经适应了西装检测和定量γH2AX灶<em&gt;体外</em&gt;<em&gt;在体内</em<sup&gt; 4,5</sup&gt;。在这里，我们展示了一个典型的免疫荧光法在小鼠组织中γH2AX灶的检测和定量。

Watch this Scientific Journal Video about Quantitation of Î³H2AX Foci in Tissue Samples at JoVE.com

Quantitation of &#947;H2AX Foci in Tissue Samples

定量DNA双链上的γH2AX灶的基础上休息，已经成为一个宝贵的工具，尤其是在放射生物学，组织的放射敏感性和辐射修改化合物的影响的评价。在这里，我们表现出的组织样本中γH2AX灶的定量免疫荧光法的使用。

定量组织样本中的γH2AX灶

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

quantitation-of-h2ax-foci-in-tissue-samples

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Quantitation of &gamma;H2AX Foci in Tissue Samples

14.3K 次观看.  The Alfred Medical Research and Education Precinct. 定量DNA双链上的γH2AX灶的基础上休息，已经成为一个宝贵的工具，尤其是在放射生物学，组织的放射敏感性和辐射修改化合物的影响的评价。在这里，我们表现出的组织样本中γH2AX灶的定量免疫荧光法的使用。

视频: Quantitation of γH2AX Foci in Tissue Samples

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 ...

科研

癌症研究

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

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

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 ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

细胞周期特异性测量βH2AX和细胞凋亡后热源毒性应力通过流细胞测定

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

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 ...

An Automated Microscopic Scoring Method for the &#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects induced by exposure to ionizing radiation to make an individual dose assessment. This is pertinent in the framework of radiation emergencies, where health assessments and planning of clinical treatment for exposed victims are critical. Since DNA double strand breaks (DSB) are considered to be the most lethal form of radiation-induced DNA damage, this protocol presents a method to detect DNA DSB in blood samples. The methodology is based on the detection of a fluorescent labelled phosphorylated DNA repair protein, namely, &#947;-H2AX. The use of an automated microscopy platform to score the number of &#947;-H2AX foci per cell allows a standardized analysis with a significant decrease in the turn-around time. Therefore, the &#947;-H2AX assay has the potential to be one of the fastest and sensitive assays for biological dosimetry. In this protocol, whole blood samples from healthy adult volunteers will be processed and irradiated in vitro in order to illustrate the usage of the automated and sensitive &#947;-H2AX foci assay for biodosimetry applications. An automated slide scanning system and analysis platform with an integrated fluorescence microscope is used, which allows the fast, automatic scoring of DNA DSB with a reduced degree of bias.

This protocol presents the slide preparation and automated scoring of the &#947;-H2AX foci assay on peripheral blood lymphocytes. To illustrate the method and sensitivity of the assay, isolated lymphocytes were irradiated in vitro. This automated method of DNA DSB detection is useful for fast and high-throughput biological dosimetry applications.

一种人外周血淋巴细胞γ-H2AX病灶测定的自动显微镜评分方法

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects ...

Dual Immunofluorescence of &#947;H2AX and 53BP1 in Human Peripheral Lymphocytes

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, including cancer. The cell is, therefore, provided with complex mechanisms to repair DSBs, and these pathways involve histone H2AX in its phosphorylated form at Ser-139 (namely &#947;H2AX) and p53 binding protein 1 (53BP1). As both proteins can form foci at the sites of DSBs, identification of these markers is considered a suitable method to study both DSBs and their kinetics of repair. According to the molecular processes that lead to the formation of &#947;H2AX and 53BP1 foci, it could be more useful to investigate their co-localization near the DSBs in order to set up an alternative approach that allows quantifying DSBs by the simultaneous detection of two DNA damage markers. Thus, this protocol aims to assess the genomic damage induced in human lymphocytes by the radiomimetic agent bleomycin through the presence of &#947;H2AX and 53BP1 foci in a dual immunofluorescence. Using this methodology, we also delineated the variation in the number of &#947;H2AX and 53BP1 foci over time, as a preliminary attempt to study the repair kinetics of bleomycin-induced DSBs.

This protocol presents a method to assess the formation and repair of DNA double-strand breaks through the simultaneous detection of &#947;H2AX and 53BP1 foci in interphase nuclei of bleomycin-treated human peripheral lymphocytes.

人外周淋巴细胞中γH2AX和53BP1的双重免疫荧光

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, ...

遗传学

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

电离辐射的反应定量的γH2AX灶

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

生物学

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

评价以下电离辐射的空间分布的γH2AX

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

技术诱导和量化细胞衰老

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.