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）。这项工作是由“儿童权利公约”，生物医学成像发展有限公司，建立和下的澳大利亚政府S合作研究中心（CRC）计划的支持。 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。

细胞制备<ol><li>人红白血病K562细胞生长在10％（V / V）饱和湿度5％的CO 2环境中的胎牛血清和20毫克/毫升的庆大霉素的RPMI - 1640补充介质在37 ° C。 </li><li>染色前约18小时，洗指数生长的细胞（5 × 10 5细胞/毫升是最优的）用磷酸盐缓冲液（PBS），悬浮在新媒体和返回到37 ° C，5％的CO 2。 </li><li>两次用PBS离心洗涤细胞，在5分钟的转速约1500，并重新悬浮在新鲜的媒体。 </li><li>计数细胞并调整细胞密度约为5 × 10 5细胞/ ml。 </li></ol>细胞可以算作一个自动化的方法（如过滤器的大小5至20微米之间的希森美康或库尔特计数器），或通过台盼蓝排除方法和血球。过量的细胞数（&gt; 800细胞/厘米2），可能会导致非均匀染色。 辐照和免疫荧光染色<ol><li> cytospin剪辑，过滤器卡和幻灯片的组装与适当的标签。 </li></ol>当装配cytospin仪器，确保漏斗过滤卡孔重合。 <ol start="2"><li>使细胞或γ-辐射或X射线（在这个例子中2Gy）。 </li></ol>为了防止在照射病灶的形成，为5至10分钟之前和期间照射细胞置于冰上。 以下照射细胞在37℃，5％的CO 2所需的时间（峰γH2AX水平的30分钟至1小时，在这个例子中的1小时）。 <ol start="3"><li>用冷PBS清洗细胞两次离心悬浮在新鲜PBS。 </li><li>在5分钟500转100到150μL的细胞悬液，免除到每个cytospin漏斗和自旋。 </li><li>从cytospins分隔的幻灯片，让细胞适度空气干燥约15分钟。 </li></ol>不要让细胞完全干燥细胞形态可能瓦解。 <ol start="6"><li>周围的细胞具有疏水笔绘制一个圆。 </li><li> 4％多聚甲醛在室温（RT）5分钟，修复细胞。 </li></ol>根据我们的经验，与多聚甲醛固定是优于乙醇或甲醇固定。悬浮细胞需要固定在室温下5分钟，而贴壁细胞的最佳固定需要较长的固定时间（15至20分钟）。 我们发现，染色立即产生更好的效果，比储存在-20 ° C，通常在高背景荧光固定的幻灯片。 <ol start="8"><li>用PBS清洗细胞两次，每次5分钟。 </li><li>通透细胞，用0.1％TRITON - X的RT 100 5分钟。 </li></ol>吐温80相比，我们通常观察与Triton X - 100通透细胞更好的信号。根据细胞类型，它可能需要改变的permeabilisation时间。例如，在K562细胞在室温下5分钟的通透性是足够的，而在15分钟的贴壁细胞通透需要。 <ol start="10"><li>洗涤细胞用PBS三次，每次5分钟。 </li><li>座在室温为30分钟（3 × 10分钟）的1％BSA的细胞。 </li></ol>我们比较使用血清（二级抗体的同一品种产生）和牛血清白蛋白的PBS阻断效率。 BSA是在最大限度地减少非特异性二次抗体结合更有效率相比，血清。我们比较了不同的阻断时间，一夜之间堵在4 ° C相比，3 × 10分钟和3 × 20分钟的阻塞在室温下的步骤，并确定为3 × 10分钟的阻塞是最佳的。 <ol start="12"><li>每张幻灯片添加50μL初级抗体（1：500 1％BSA稀释）。 </li></ol>孵育过夜抗体在室温为60分钟，结果减少背景染色的潜伏期比4 ° C。 <ol start="13"><li>染色不同的标记，同时使用在不同的物种（如鼠抗γH2AX和兔抗在这个例子中的甲基化H3K4）提出的抗体。 </li><li>在室温为90分钟，在250 RPM摇摆平台上小学抗体孵育。 </li></ol>所有孵化加湿染色槽。 <ol start="15"><li>洗涤细胞用PBS三次，每次5分钟。 </li><li>加入50μL（1：1000稀释在1％BSA）二级抗体，以每张幻灯片。 </li></ol>染色同一张幻灯片上的不同标记，使用二次抗体与Different种属特异性（抗体）和荧光发射波长（如抗鼠Alexa的488（绿色）和抗兔Alexa的- 546（红色）在这个例子中）。 <ol start="17"><li>在室温为60分钟，在250 RPM摇摆平台上培养的细胞。 </li></ol>建议在黑暗潮湿的会议厅，避免褪色和干燥的二级抗体孵育的细胞。 <ol start="18"><li>洗涤细胞用PBS三次，每次5分钟。 </li><li>每张幻灯片添加50μL（1:500 PBS稀释）TOPRO - 3。 </li><li> 3X 5分钟，用PBS冲洗幻灯片。 </li><li>从幻灯片中删除多余的水分和添加防褪色的解决方案。 </li></ol>这是建议，没有完全晾干后方可加入防褪色的解决方案的细胞。 <ol start="22"><li>盖玻片，指甲油密封和离开幻灯片在黑暗中过夜，在室温。 </li></ol>这是推荐使用的具体盖玻片焦＃1.5（0.16-19微米厚）和透明指甲油。 在幻灯片上放置盖玻片，应小心，以避免形成气泡。 图像采集<ol><li>蔡司LSM510元共聚焦显微镜是用来获取图像，使用标准GFP（绿色，488纳米），PI（红色543纳米）和远红光（蓝色633纳米）的激光。 </li></ol>图片收购，使用共聚焦显微镜，揭示了更好的空间分辨率。 由于γH2AX灶的大小可能会低于0.5μicrons，它是建议获得至少0.5μicron沿Z轴的步长大小的图像。 一个63 ×油浸物镜相比或高或低放大倍率是首选。对于成像许多灶计数细胞，最好是使用8个扫描速度和图像尺寸1024 × 1024像素。为了说明不同，它是建议使用2048 × 2048像素图像的大小为6扫描速度的核蛋白质的空间之间的关系。 当来自多个渠道的成像相比，一帧扫描线扫描收购建议。这就避免了漂白和更好的分辨率达到。顺序扫描是用来防止渠道之间的流血。 佛慈计数图像分析和灶计数，可以使用各种套装软件（计有Metamorph，图像- J和Imaris）。这里描述灶使用Metamorph计数程序。 <ol><li>无论是直接从文件菜单，或通过建立数堆栈选项打开γH2AX形象栈。 </li><li>转到进程“菜单，并选择堆栈算术。 </li><li>选择了最大的投影，选择要包含的飞机，并单击“确定”。 </li><li>保存作为γH2AXTIFF图像。文件，并打开TOPRO - 3（蓝色）的图像。 </li><li>进入的地区，选择一个区域的绘图工具画原子核周围的地区。 </li><li> γH2AXTIFF图像处理菜单，并选择形态滤波器。 </li><li>选择顶帽子，并选择γH2AX图像作为源图像。 </li><li>应用自顶帽子过滤器和由此产生的图像将是一个二进制图像灶强度，低噪音和减少变异。 </li></ol>顶帽子值时，应注意选择。最优值，可以得到图像的视觉对比前后应用过滤器。 <ol start="9"><li>前往处理菜单，并选择阈值图像。 </li><li>选择包容性阈值和选择较低和较高的阈值。 </li></ol>它通常是影响癌灶数目的阈值。因此，选择阈值时，需要十分重视。这可以最好地实现计数灶使用不同的阈值值在细胞暴露于γ辐射低于2 Gy的，然后比较与人工计数（眼），疫源地号码。最优阈值是一个最小化背景的包容性和最大限度地提高列入灶。 <ol start="11"><li>选择TOPRO - 3的形象和地区菜单，并选择转移地区选项。选择TOPRPO - 3的源图像和顶帽子的目标，并单击确定。 </li><li>一旦核地区转移到了顶帽子的形象去测量菜单，并选择综合形态计量学分析。 </li><li>在弹出窗口中选择源图像顶帽子和按面积的措施。 </li><li>选择测量上衡量各地区，各获得所有相应的核顶帽子的形象灶数量。 </li><li>疫源地从每个区域的数字可以被导出到一个MS - Excel文件，使用日志数据的选项。 </li></ol> 3D图像重建要创建一个三维图像，从使用Metamorph栈： <ol><li>转到堆栈菜单选择三维重建。 </li><li>选择旋转的角度（如160 °，320 °）。 </li><li>选择类型最大的三维重建。 </li><li>选择旋转平面（水平或垂直）。 </li><li>选择的Z -校准距离。 </li></ol>最好是使用用户指定的Z轴的距离（最佳为0.5μicrons）。 <ol start="6"><li>选择确定以创建一个三维重建。 </li></ol>线扫描分析对于线扫描分析，使用Metamorph： <ol><li>打开一个单一的平面图像或堆放文件。 </li><li>转到测量工具栏“命令，选择线扫描分​​析。 </li><li>整个单元格中画一条线，这将自动显示每个标记的荧光强度和距离沿行的路径。 </li></ol>为了澄清各种染色标记的空间关系，例如，γH2AX灶和组蛋白甲基化，这是可取的画线，跨越地区，都是密集的差，在这些标记。 具有代表性的数据对于这个演示的目的，我们利用抗体来γH2AX灶和H3K4me，双链断裂的空间分布来评价一个积极抄写常染色质的表观因素。如图1所示，与2Gy照射后γH2AX灶形成，主要为H3K4me染色和DNA染色TOPRO - 3（鲜艳染色的DNA区域指示异）沉闷的地区。 <img src="/files/ftp_upload/2203/2203fig1.jpg" alt="图1" /> 图1。γH2AX灶形式在常染色质主要在电离辐射。 （一）人红白血病K562细胞的免疫可视化γH2AX灶（绿色），1小时后，γ射线照射（2戈瑞）。 γH2AX灶有关H3K4me，代表积极抄写常染色质（红色）。 DNA标记TOPRO - 3（蓝色）。合并后的图像（蓝色，红色和绿色）演示了从排斥异色地区γH2AX灶。线扫描分析（荧光强度与距离）表示在一个平面上的标记相对分布。 （二）在不同的飞机从合并后的图像三维重建片上面所述。

<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 (5), 905-916 (1999).</li><li>Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S., 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=DNA+double-stranded+breaks+induce+histone+H2AX+phosphorylation+on+serine+139.">DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.</a> J Biol Chem. 273 (10), 5858-5868 (1998).</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=.">.</a> Nat Rev 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>Kim, J. A., Kruhlak, M., Dotiwala, F., Nussenzweig, A., Haber, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochromatin+is+refractory+to+gamma-H2AX+modification+in+yeast+and+mammals.">Heterochromatin is refractory to gamma-H2AX modification in yeast and mammals.</a> J Cell Biol. 178 (2), 209-218 (2007).</li><li>Cowell, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gammaH2AX+foci+form+preferentially+in+euchromatin+after+ionising-radiation.">gammaH2AX foci form preferentially in euchromatin after ionising-radiation.</a> PLoS One. 2 (10), e1057-e1057 (2007).</li><li>Kinner, A., Wu, W., Staudt, C., &amp;amp, I. l. i. a. k. i. s., G, . <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+in+recognition+and+signaling+of+DNA+double-strand+breaks+in+the+context+of+chromatin.">Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin.</a> Nucleic Acids Res. 36 (17), 5678-5694 (2008).</li><li>Vasireddy, R. S., Karagiannis, T. C., El-Osta, 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-radiation-induced+gammaH2AX+formation+occurs+preferentially+in+actively+transcribing+euchromatic+loci.">gamma-radiation-induced gammaH2AX formation occurs preferentially in actively transcribing euchromatic loci.</a> Cell Mol Life Sci. 67 (2), 291-294 (2010).</li><li>Goodarzi, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATM+signaling+facilitates+repair+of+DNA+double-strand+breaks+associated+with+heterochromatin.">ATM signaling facilitates repair of DNA double-strand breaks associated with heterochromatin.</a> Mol Cell. 31 (2), 167-177 (2008).</li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Roswell Park Memorial Institute -1640 (RPMI-1640)</td>
<td>Growth medium</td>
<td>Invitrogen</td>
<td>22400071</td>
<td>RPMI-1640, pH 7.4 medium supplemented with 10% (v/v) fetal bovine, 2mM L-glutamine, 20&#x03BC;g/ml gentamicin, 20mM (HEPES) N-2-Hydroxyethylpiperazine-N&#x2019;-2-Ethanesulfonic Acid</td>
</tr>
<tr>
<td>Fetal Bovine Serum (FBS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F2442</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin (BSA)</td>
<td>&nbsp;</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 Ca2+ and Mg2+)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17-517Q</td>
<td>&nbsp;</td>
<tr>
<td>Trypan blue</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>T6146</td>
<td>Used to distinguish between live and dead 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>Paraformaldehyde (4%) used to fix cells.</td>
</tr>
<tr>
<td>Mouse monoclonal anti-phospho histone-H2AX antibody</td>
<td>Primary Antibody</td>
<td>Millipore, USA</td>
<td>16193</td>
<td>Dilution of primary antibody (1:500), in 1% BSA.</td>
</tr>
<tr>
<td>HistoneH3 (Mono-methyl K4) </td>
<td>Primary Antibody</td>
<td>Abcam, Cambridge, UK</td>
<td>AB8895</td>
<td>&nbsp;</td>
<tr>
<td>Alexa Fluor 488 goat anti-mouse IgG (H+L)</td>
<td>Secondary Antibody</td>
<td>Invitrogen, USA</td>
<td>11029</td>
<td>Dilution of secondary antibody (1:500), in 1% BSA.</td>
</tr>
<tr>
<td>Alexa Fluor 546 goat anti-rabbit IgG (H+L)</td>
<td>Secondary Antibody</td>
<td>Invitrogen, USA</td>
<td>11035</td>
<td>&nbsp;</td>
<tr>
<td>TOPRO3</td>
<td>DNA Stain</td>
<td>Invitrogen, USA</td>
<td>T3605</td>
<td>TOPRO3 is a DNA dye with an Abs/Em of 642/661 nm. DAPI could be used if the confocal microscope is equipped with a 405 nm laser. </td>
</tr>
<tr>
<td>ProLong Gold </td>
<td>Anti-fade solution</td>
<td>Invitrogen</td>
<td>P36930</td>
<td>This glycerol based mounting medium must be used with an oil based lense that matches its refractive index.</td>
</tr>
<tr>
<td>Polylysine slides</td>
<td>&nbsp;</td>
<td>Menzel-Gl&#228;ser, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (22x50mm)</td>
<td>Coverslips</td>
<td>Menzel-Gl&#228;ser, Germany</td>
<td>CS2250100</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Culture Flask, Vented Cap</td>
<td>Culture Flask</td>
<td>BD Falcon</td>
<td>353112</td>
<td>&nbsp;</td>
<tr>
<td>Shandon Cytospin 4</td>
<td>&nbsp;</td>
<td>ThermoElectron Corp</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cytofunnels</td>
<td>&nbsp;</td>
<td>Shandon, Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Filter Cards </td>
<td>&nbsp;</td>
<td>Shandon, Inc</td>
<td>353025</td>
<td>&nbsp;</td>
<tr>
<td>Coplin Jar, glass</td>
<td>&nbsp;</td>
<td>Grale Scientific P/L</td>
<td>1771-OG</td>
<td>&nbsp;</td>
<tr>
<td>Staining Trough</td>
<td>&nbsp;</td>
<td>Grale Scientific P/L</td>
<td>V1991.99</td>
<td>&nbsp;</td>
<tr>
<td>PAP Pen</td>
<td>&nbsp;</td>
<td>Zymed Laboratories, Invitrogen</td>
<td> 008877</td>
<td>&nbsp;</td>
<tr>
<td>Gammacell 1000 Elite Irradiator</td>
<td>Gamma Irradiator</td>
<td>Nordion International Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Zeiss LSM 510 Meta Confocal</td>
<td>Confocal Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Equipped with 3 lasers: 488 nm, 543 nm and 633 nm.</td>
</tr>
<tr>
<td>Metamorph</td>
<td>Software for Imaging analysis</td>
<td>Molecular Devices, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

evaluation of the spatial distribution of &gamma;h2ax following ionizing radiation

早期的分子反应，DNA双链断裂（DSB的）磷酸化的SER - 139内的组蛋白H2AX 1,2终端SQEY图案的残留。 H2AX的磷酸化介导的磷脂，inosito 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，ATM和相关RAD3（ATR）的3。 H2AX的磷酸化形式，简称为γH2AX，蔓延到邻近地区的染色质的DSB网站，形成离散灶，很容易由3 immunofluorecence显微镜可视化。分析和定量γH2AX灶已被广泛用于评估DSB的形成和修复，特别是在电离辐射和评估各种修改化合物和细胞毒素化合物4辐射的功效， 。 鉴于这种双链断裂从头标志的精美的特异性和灵敏度，它在染色质中的DNA损伤和修复的过程提供新的见解。例如，在辐射生物学中心的范例是核DNA与放射敏感性的关键目标。事实上，在该领域的普遍共识在很大程度上被认为染色均匀的模板为DNA损伤与修复。然而，与γH2AX作为分子标记，DNA双链断裂，在γ射线照射，诱导γH2AX灶在常染色质和异染色质的形成悬殊的使用已被观察到5-7。最近，我们使用的抗体面板，无论是单，双或三甲基化组蛋白H3赖氨酸9（H3K9me1，H3K9me2，H3K9me3）后生印记构异染色质和转录沉默和赖氨酸4（H3K4me1，H3K4me2，H3K4me3 ），这是密切相关的积极抄写常染色质区域，调查γH2AX的空间分布， 电离辐射8。按照当时的想法，关于染色质生物学，我们的研究结果表明γH2AX的形成和活跃转录9之间的密切相关。在这里，我们展示了非贴壁细胞中γH2AX灶的检测和定量的免疫方法，特别注重与其他的表观遗传标记，图像分析和三维建模与合作本地化。

Watch this Scientific Journal Video about Evaluation of the Spatial Distribution of Î³H2AX following Ionizing Radiation at JoVE.com

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

微观分析γH2AX灶，形成以下丝氨酸139磷酸化H2AX在DNA双链断裂，已成为辐射生物学中的一个非常宝贵的工具。在这里，我们使用一种抗体，单甲基化的组蛋白H3赖氨酸4作为一个积极抄写常染色质的表观遗传标记，以评估辐射诱导γH2AX形成的细胞核内的空间分布。

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 ...

evaluation-spatial-distribution-h2ax-following-ionizing

Research

JoVE Journal

Biology

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

12.6K 次观看.  The Alfred Medical Research and Education Precinct. 微观分析γH2AX灶，形成以下丝氨酸139磷酸化H2AX在DNA双链断裂，已成为辐射生物学中的一个非常宝贵的工具。在这里，我们使用一种抗体，单甲基化的组蛋白H3赖氨酸4作为一个积极抄写常染色质的表观遗传标记，以评估辐射诱导γH2AX形成的细胞核内的空间分布。

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

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 &amp;#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 ...

科研

生物学

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

生产 C。线虫</em通过重组工程与转基因 galK选择标记

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

切片培养法继牙胚在植文化的发展

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.

SCAP分析<em&gt;ñ</em&gt; -glycosylation和贩卖人体细胞

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of ...

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro and in vivo. Fluorescent probes are particularly useful in determining the bio-distribution of administered large molecules, where the addition of a small-molecule fluorescent label is unlikely to affect the kinetics or bio-distribution of the compound. A variety of methods exist to examine bio-distribution that vary significantly in the amount of effort required and whether the resulting measurements are fully quantitative, but using multiple methods in conjunction can provide a rapid and effective system for analyzing bio-distributions.
Ex vivo whole-organ imaging is a method that can be used to quickly compare the relative concentrations of fluorescent molecules within tissues and between multiple types of tissues or treatment groups. Using an imaging platform designed for live-animal or whole-organ imaging, fluorescence within intact tissues can be determined without further processing, saving time and labor while providing an accurate picture of the overall bio-distribution. This process is ideal in experiments attempting to determine the tissue specificity of a compound or for the comparison of multiple different compounds. Quantitative tissue histology on the other hand requires extensive further processing of tissues in order to create a quantitative measure of the labeled compounds. To accurately assess bio-distribution, all tissues of interest must be sliced, scanned, and analyzed relative to standard curves in order to make comparisons between tissues or groups. Quantitative tissue histology is the gold standard for determining absolute compound concentrations within tissues.
Here, we describe how both methods can be used together effectively to assess the ability of different administration methods and compound modifications to target and deliver fluorescently labeled molecules to the central nervous system1.

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Ex vivo whole-organ imaging is a rapid method for determining the relative concentrations of fluorescently labeled compounds within and between tissues or treatment groups. Conversely, quantitative fluorescence histology, while more labor intensive, allows for the quantification of the absolute tissue levels of labeled molecules.

指某东西的用途<em&gt;离体</em&gt;全器官成像和定量组织组织学确定荧光标记分子的生物分布

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

尼罗红与油红 O 染色法测定秀丽线虫的脂质丰度及脂质分布评价

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose determination using low energy X-ray facilities, but depending on the irradiation configurations, samples, materials or beam quality, it is sometimes difficult to know which protocol is the most appropriate to employ. We, therefore, propose a dosimetry protocol for cell irradiations using low energy X-ray facility. The aim of this method is to perform the dose estimation at the level of the cell monolayer to make it as close as possible to real cell irradiation conditions. The different steps of the protocol are as follows: determination of the irradiation parameters (high voltage, intensity, cell container etc.), determination of the beam quality index (high voltage-half value layer couple), dose rate measurement with ionization chamber calibrated in air kerma conditions, quantification of the attenuation and scattering of the cell culture medium with EBT3 radiochromic films, and determination of the dose rate at the cellular level. This methodology must be performed for each new cell irradiation configuration as the modification of only one parameter can strongly impact the real dose deposition at the level of the cell monolayer, particularly involving low energy X-rays.

Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

This document describes a new dosimetry protocol for cell irradiations using low energy X-ray equipment. Measurements are performed in conditions simulating real cell irradiation conditions as much as possible.

使用矫形器（40-300 kV）X射线设施进行细胞辐照的测定

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose ...

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H2O2), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1&#946;, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-&#945;) assay, and cells were evaluated for viability using fluorescent dyes. 
The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H2O2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line.

确定紫外线辐射和化学物质对原代和永生化的人角膜上皮细胞的毒性

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...