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。

<html>细胞制备<ol><li>生长在角质形成细胞无血清培养人角质形成细胞（FEP - 1811）（K - SFM; Invitrogen公司）与表皮生长因子，牛垂体提取物和20微克/毫升庆大霉素的补充，在37 ° C和5％的CO 2 。 </li><li>用胰酶- EDTA（0.05％V / V）分离制备单细胞悬液</li><li>细胞接种于8实验室TEK第二microchamber幻灯片（万细胞/孔）和幻灯片，在37 ° C和5％的CO 2孵育3天。 </li></ol>放射<ol><li>冰使用的137 Cs源（1000 Gammacell精英辐照; Nordion公司国际上，加拿大; 20.6秒/ GY）与2戈瑞辐照细胞</li><li>未经辐照的控制和2 Gy照射细胞孵育1小时37 ° C和5％的CO 2 。 </li></ol>免疫荧光染色<ol><li>媒体通风报信，洗涤细胞，每孔300μL的PBS（W / O CA 2 +或Mg 2 +的 ），并在5分钟的轨道混频器的旋转。 </li><li>缓冲区是通风报信，新鲜配制的4％多聚甲醛（V / V），每孔加入幻灯片在室温下孵育10分钟100μL。 所有孵化加湿染色槽</li><li>细胞，然后用PBS（W / O型的Ca 2 +，Mg 2 +的 ）。 5分钟的轨道混频器，幻灯片被放置在一个Coplin JAR和旋转。此清洗步骤再重复两次。 </li><li>缓冲区是通风报信，多余的PBS轻轻涂抹。 </li><li>使用100毫升TRITON X - 100（0.1％V / V），每口井，在室温下孵育10分钟的细胞透。 </li><li>细胞，用PBS冲洗（W / O型的Ca 2 +或Mg 2 +的 ）如在上述步骤3和4中所述</li><li>在室温下（1％V / V）与BSA的100毫升的非特异性蛋白结合，阻止每口井和20分钟的孵育。 </li><li>多余的BSA是通风报信，鼠抗磷酸化组蛋白H2AX的抗体（1:500稀释，在1％BSA; Millipore公司）100μL，在室温下孵育1小时，每口井。 </li><li>细胞用PBS冲洗（W / O型的Ca 2 +或Mg 2 +）的步骤3和第4段中所述和培养100μL二次抗体（的Alexa Fluor 488山羊抗鼠IgG稀释1:500，在1％BSA ; Invitrogen公司），每45分钟，在室温下在黑暗中。 （摊薄抗体，整个过程被蒙在鼓里） </li><li>细胞，用PBS冲洗（W / O型的Ca 2 +或Mg 2 +的 ）如在上述步骤3和4中所述然而，暴露在光线下是最小化使用铝箔。 </li><li>核counterstaining是用100ml TOPRO3（稀释1:500; Invitrogen公司）进行，每口井，在室温下孵育10分钟的</li><li>细胞用PBS（W / O型的Ca 2 +或Mg 2 +）在第10步以上所述。 </li><li>该商会进行了仔细从幻灯片中删除多余的水分，涂抹和幻灯片被允许空气干燥。 </li><li>延长金防褪色的解决方案（Invitrogen）的下降，每孔加入幻灯片安装（22x50毫米盖玻片）和幻灯片的边缘周围的任何多余的液体被抹杀。 </li><li>被关在黑暗为进一步在室温下30分钟，然后用指甲油密封幻灯片。 </li><li>幻灯片一夜之间被储存在4 ° C的分析前的黑暗。 </li></ol>显微镜/分析<ol><li>蔡司LSM510 META共焦Microscpe用来获取图像，使用标准的绿色荧光蛋白（γH2AX - 的Alexa Fluor 488山羊抗鼠IgG）和远红光激光器（TOPRO - 3）。通常，一个63 ×油浸物镜使用。 一个0.5微米的一步大小，图像采集在Z系列模式。一个0.5微米的一步大小选择不同的平面，以尽量减少损失灶目前在细胞核。在分析过程中，个别飞机卷积和堆放产生一个最大投射影像的疫源地，以尽量减少重叠（顶帽子过滤器适用于）。 </li><li> Metamorph分子器件，（美国）是用来分析疫源地的数量。 该计划定量灶，在每个单元的阈值后，已被应用到排除的背景。该信息被记录在Microsoft Excel电子表格，作进一步的分析。 </li></ol><img src="/files/ftp_upload/1957/1957fig1.jpg" alt="图1" /> 图1。γH2AX灶（绿色）的免疫可视化未经处理的人角质形成细胞和细胞在2 Gy的照射，并为进一步的1小时在37℃，5％的CO 2。 DNA染色TOPRO - 3（蓝色）。使用的Zeiss LSM 510 META激光共聚焦显微镜获得的图像。酒吧= 10微米。 e_content“&gt; <img src="/files/ftp_upload/1957/1957fig2.jpg" alt="图2" /> 图2。γH2AX灶（绿色）的免疫可视化在人类角质形成细胞和细胞在2 Gy的照射，并为进一步的1小时在37℃，5％的CO 2。图片被收购使用的Zeiss LSM 510元共聚焦显微镜使用0.5微米的Z -切片（1-9），以确保所有灶被收购。的图像，然后堆放定量使用Metamorph。 DNA染色TOPRO - 3（蓝色）。堆叠堆叠γH2AX和蓝色的图像可视化。酒吧= 10微米。

<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>Savic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+Dynamic+[gamma]-H2AX+Domains+along+Broken+DNA+Strands+Is+Distinctly+Regulated+by+ATM+and+MDC1+and+Dependent+upon+H2AX+Densities+in+Chromatin.">Formation of Dynamic [gamma]-H2AX Domains along Broken DNA Strands Is Distinctly Regulated by ATM and MDC1 and Dependent upon H2AX Densities in Chromatin.</a> Molecular Cell. 34 (3), 298-310 (2009).</li><li>Downs, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+chromatin-modifying+activities+to+phosphorylated+histone+H2A+at+DNA+damage+sites.">Binding of chromatin-modifying activities to phosphorylated histone H2A at DNA damage sites.</a> Mol Cell. 16 (6), 979-990 (2004).</li><li>Chowdhury, D. <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+Dephosphorylation+by+Protein+Phosphatase+2A+Facilitates+DNA+Double-Strand+Break+Repair.">[gamma]-H2AX Dephosphorylation by Protein Phosphatase 2A Facilitates DNA Double-Strand Break Repair.</a> Molecular Cell. 20 (5), 801-809 (2005).</li><li>Nakada, S., Chen, G., Gingras, A., Durocher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PP4+is+a+gamma+H2AX+phosphatase+required+for+recovery+from+the+DNA+damage+checkpoint.">PP4 is a gamma H2AX phosphatase required for recovery from the DNA damage checkpoint.</a> EMBO Rep. 9 (10), 1019-1026 (2008).</li><li>Altaf, M., Auger, A., Covic, M., C&#244;t&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connection+between+histone+H2A+variants+and+chromatin+remodeling+complexes.">Connection between histone H2A variants and chromatin remodeling complexes.</a> Biochem Cell Biol. 87 (1), 35-50 (2009).</li><li>Kusch, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylation+by+Tip60+Is+Required+for+Selective+Histone+Variant+Exchange+at+DNA+Lesions.">Acetylation by Tip60 Is Required for Selective Histone Variant Exchange at DNA Lesions.</a> Science. 306, 2084-2087 (2004).</li><li>Hurlin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+human+papillomavirus+type+18-immortalized+human+keratinocytes+to+a+malignant+phenotype.">Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype.</a> Proc Natl Acad Sci U S A. 88 (2), 570-574 (1991).</li><li>Dickey, J. <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. , (2009).</li></ol>

暴露于电离辐射（γ射线），γH2AX灶形式迅速和疫源地号码后达到最大，在30-60 分钟之间2。因此，1小时后照射时间点反映初始DSB的形成。我们的实验中，我们已经用2 Gy的临床相关的辐射剂量。然而，该方法可用于辐射剂量高达4戈瑞检测初始DSB的形成;灶明显的重叠排除在较高剂量准确定量。可用于更高的辐射剂量辐照后的潜伏期较长时间，γH2AX灶因维修，造成量化的数字。通常情况下，4...

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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>Keratinocyte-Serum Free Medium (K-SFM)</td>
<td>Media</td>
<td>Invitrogen</td>
<td>17005042</td>
<td>Keratinocyte media supplemented with human recombinant Epidermal Growth Factor 1-53 (EGF 1-53), and Bovine Pituitary Extract (BPE).</td>
</tr>
<tr>
<td>Lab Tek lI Chamber Slides (8-well)</td>
<td>Chamber Slides</td>
<td>Nunc Denmark</td>
<td>NUN154534</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (22x50mm)</td>
<td>Coverslips</td>
<td>Menzel-Gl&auml;ser</td>
<td>CS2250100</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>0.5% Trypsin-EDTA x10</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15400-054</td>
<td>Trypsin-EDTA (0.05%) used to detach cells from culture flasks.</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</td>
<td>16193</td>
<td>Dilution of primary antibody (1:500), in 1% BSA.</td>
</tr>
<tr>
<td>Alexa Fluor 488 goat anti-mouse IgG (H+L)</td>
<td>Secondary Antibody</td>
<td>Invitrogen</td>
<td>11029</td>
<td>Dilution of secondary antibody (1:500), in 1% BSA.</td>
</tr>
<tr>
<td>TOPRO3</td>
<td>DNA Stain</td>
<td>Invitrogen</td>
<td>T3605</td>
<td>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>ProLong Gold</td>
<td>Anti-fade solution</td>
<td>Invitrogen</td>
<td>P36930</td>
<td>Refractive index of 1.42 at 20&deg;C.</td>
</tr>
<tr>
<td>Tissue Culture Flask, Vented Cap</td>
<td>Culture Flask</td>
<td>BD Falcon</td>
<td>353112</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Culture Dish (150x25mm)</td>
<td>Petridish</td>
<td>BD Falcon</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>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>&nbsp;</td>
<tr>
<td>Metamorph</td>
<td>Software for Imaging analysis</td>
<td>Molecular Devices, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

quantification of &gamma;h2ax foci in response to ionising radiation

DNA双链断裂（DSB的），这是无论是内源性代谢过程或外源性​​诱导，是最关键的DNA损伤一个方面的生存和保持基因组完整性。早期反应的双链断裂感应是组蛋白H2A变体，H2AX的磷酸化，丝氨酸139残留，在高度保守的C -末端SQEY图案，形成γH2AX<sup&gt; 1</sup&gt;。 （ATR）的双链断裂感应后，H2AX的迅速磷酸化的磷脂，inosito 3 - 激酶（PIKK）蛋白家族，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<sup&gt; 2</sup&gt;。通常情况下，只有少数几个碱基对（BP）在DSB有牵连，但是，有显着的信号放大，信号和修复DNA损伤的染色质修饰的重要性。 H2AX的磷酸化介导主要由ATM染色邻近地区的利差，影响约0.03％的细胞总H2AX的每DSB<sup&gt; 2,3</sup&gt;。这相当于约2000 H2AX的分子跨越〜2 MBP染色质的地区，周围的DSB网站和离散γH2AX灶形成的结果，可以很容易地可视化和免疫荧光显微镜定量磷酸化<sup&gt; 2</sup&gt;。 γH2AX在DSB的损失反映了修复，但是，有一些争论什么定义完整的双链断裂的修复，它已经提出，重新加入DNA的两条链是足够的，然而，它也被建议重新恢复压实原有的染色质状态是必要的<sup&gt; 4-8</sup&gt;。 γH2AXdisappearence涉及至少部分，去磷酸化磷酸酶，磷酸酶2A和磷酸4C<sup&gt; 5,6</sup&gt;。此外，去除再分配涉及与H2A.Z组蛋白交流的γH2AX有牵连<sup&gt; 7,8</sup&gt;。重要的是，γH2AX灶的定量分析，导致了广泛的应用在医学和核研究。在这里，我们展示了初始γ辐照壁人角质形成细胞DNA的损伤γH2AX灶的检测和定量的评价最常用的免疫方法<sup&gt; 9</sup&gt;。

Watch this Scientific Journal Video about Quantification of ÃŽÂ³H2AX Foci in Response to Ionising Radiation at JoVE.com

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

DNA双链分子标记γH2AX形成条纹的量化已成为辐射生物学中的一个非常宝贵的工具。在这里，我们表现出免疫荧光法检测细胞对辐射的暴露后γH2AX灶的定量使用。

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 ...

quantification-of-h2ax-foci-in-response-to-ionising-radiation

Research

JoVE Journal

Biology

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

20.3K 次观看.  The Alfred Medical Research and Education Precinct. DNA双链分子标记γH2AX形成条纹的量化已成为辐射生物学中的一个非常宝贵的工具。在这里，我们表现出免疫荧光法检测细胞对辐射的暴露后γH2AX灶的定量使用。

视频: Quantification of γH2AX Foci in Response to Ionising Radiation

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event 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)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

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) ...

科研

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 ...

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic development 1, 2. One of the signaling pathways involved in this regulation is the Wnt pathway, which is used many times during embryogenesis and critical for human disease3, 4, 5. Multiple molecular components of this pathway coordinately regulate signaling in a spatially-restricted manner, but the underlying mechanisms are not fully understood. Xenopus embryonic epithelial cells is an excellent system to study subcellular localization of various signaling proteins. Fluorescent fusion proteins are expressed in Xenopus embryos by RNA microinjection, ectodermal explants are prepared and protein localization is evaluated by epifluorescence. In this experimental protocol we describe how subcellular localization of Diversin, a cytoplasmic protein that has been implicated in signaling and cell polarity determination6, 7 is visualized in Xenopus ectodermal cells to study Wnt signal transduction8. Coexpression of a Wnt ligand or a Frizzled receptor alters the distribution of Diversin fused with red fluorescent protein, RFP, and recruits it to the cell membrane in a polarized fashion 8, 9. This ex vivo protocol should be a useful addition to in vitro studies of cultured mammalian cells, in which spatial control of signaling differs from that of the intact tissue and is much more difficult to analyze.

Polarized Translocation of Fluorescent Proteins in Xenopus Ectoderm in Response to Wnt Signaling

Xenopus embryonic ectoderm has become an attractive model for studies of cell polarity. An assay is described, in which subcellular distribution of fluorescent proteins is assessed in ectoderm cells. This protocol will help address questions related to spatial control of signaling.

偏光易位荧光蛋白爪蟾外胚层响应Wnt信号

Cell polarity is a fundamental property of eukaryotic cells that is dynamically regulated by both intrinsic and extrinsic factors during embryonic ...

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 ...

Quantification of Orofacial Phenotypes in Xenopus

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial development will allow for more rigorous analysis of orofacial phenotypes upon abrogation with substances that can genetically or molecularly manipulate gene expression or protein function. Using two dimensional images of the embryonic heads, traditional size dimensions-such as orofacial width, height and area- are measured. In addition, a roundness measure of the embryonic mouth opening is used to describe the shape of the mouth. Geometric morphometrics of these two dimensional images is also performed to provide a more sophisticated view of changes in the shape of the orofacial region. Landmarks are assigned to specific points in the orofacial region and coordinates are created. A principle component analysis is used to reduce landmark coordinates to principle components that then discriminate the treatment groups. These results are displayed as a scatter plot in which individuals with similar orofacial shapes cluster together. It is also useful to perform a discriminant function analysis, which statistically compares the positions of the landmarks between two treatment groups. This analysis is displayed on a transformation grid where changes in landmark position are viewed as vectors. A grid is superimposed on these vectors so that a warping pattern is displayed to show where significant landmark positions have changed. Shape changes in the discriminant function analysis are based on a statistical measure, and therefore can be evaluated by a p-value. This analysis is simple and accessible, requiring only a stereoscope and freeware software, and thus will be a valuable research and teaching resource.

Quantification of Orofacial Phenotypes in Xenopus

A method to quantify the orofacial size and shape of Xenopus laevis embryos has been developed. In this protocol, traditional size measurements are combined with geometric morphometrics to allow for more sophisticated analyses of orofacial development and defects.

口面表型的定量<em&gt;爪蟾</em

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

测量水润谷胱甘肽诱导饲养响应

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

实验方法来研究线粒体定位和核细胞周期激酶CDK1功能

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

考试蛋白在哺乳动物细胞中绑定到新生的DNA使用的BrdU芯片插槽西方技术

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

结晶纤维素积累高分辨率的量化<em&gt;拟南芥</em&gt;根监视特定组织细胞壁修改

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...