这项工作得到了医学研究理事会和玛丽居里国际重返社会补助金计划（JKV）。

1。采摘基因表达克隆<ol><li>被提供作为在甘油中的96孔格式的单个细菌克隆的cDNA文库。中的选择器菜​​单的NORGEN CP7200中使用Rearray命令，从源板和接种到1.5毫升的LB /安培在96深孔（1毫升孔）板挑细菌克隆。 </li><li>密封的深孔板有具有透气性的密封，并在摇动器在37℃下孵育过夜。 </li></ol> 2。制备cDNA表达文库<ol><li>旋转含有细菌的20分钟，使用板适配器在台式离心机以2,000 rpm的深孔板。 </li><li>吸出井，留下的细菌沉淀完整的媒体。 </li><li>配置，如在图2中所示的实验室自动化工作站甲板。解的质粒制备分布到波谷1-5中，并放置在甲板上的深孔板。多孔槽，洗脱解决方案被置于相邻的深孔板。提示装入的前端机架。 </li><li>组装真空多方面的。质粒结合板被放置在歧管和质粒的过滤板被放置在歧管的顶部。 </li><li> 250μl溶液1（重悬缓冲液）上下吹打重悬颗粒。 </li><li>裂解细菌，通过加入250微升溶液2（裂解缓冲液）。该板是密封的，并倒相，以允许完全混合。 </li><li>开始2分钟计时器。 </li><li>加入350μl溶液3（中和缓冲）。密封板和反转的3倍。 </li><li>细菌裂解液转移到质粒的过滤板。 </li><li>施加真空5分钟。 </li><li>拆下过滤器板和歧管的顶部放置的结合板。施加真空，持续1分钟。 </li><li>添加500微升额外的洗涤缓冲液（AW）。 </li><li>施加真空，持续2分钟。 </li><li>加入900μL洗涤缓冲液的解决方案4。施加真空，持续2分钟。 </li><li>重复小号TEP 2.14。应用真空额外的15分钟。 </li><li>将歧管内的DNA收集板。的洗脱缓冲液中加入100μl的结合板孵育2分钟。 </li><li>真空10分钟。 </li><li>删除收集板和测量DNA浓度的NanoDrop 8000。通常情况下，收率〜100毫微克/微升，用这种方法，可以预期。 </li></ol> 3。细胞接种<ol><li> HEK293细胞用胰蛋白酶消化并计数。 </li><li>被分配到使用赛默飞世96孔多点了珀金埃尔默Viewplate的各孔2×10 4个细胞。 </li><li>将细胞在37℃和5％CO 2的温育过夜。 </li></ol> 4。转染<ol><li>准备用100 ng的cDNA中加入25μl无血清培养基中，在圆底96孔板中，每孔为100 ng GFP-Grb2的质粒DNA混合。 </li><li>加入25μL无血清培养基含有0.5微升Transfectin的每口井。 </li><li>混合并启动定时器FOR 30分钟。 </li><li>使用一个温柔的分配方法，将50微升的基因/ Transfectin混合细胞。可替代地，转染混合物可以被分配第一成板材，然后的细胞增加到顶端的（反向转染）。 </li><li>孵育细胞过夜，在37℃和5％CO 2的 。 </li></ol> 5。图像采集<ol><li>启动歌剧院软件。实验设置的屏幕快照， 如图3所示。选择“配置”选项卡，并选择20倍的目标和正确的板型。确保“围脖”设置为正确的值目标为重点，以允许不同的板块类型。 </li><li>选择“显微镜”选项卡。定义1 488激光（FITC）和曝光2曝光UV（365激光）。激活两次曝光的紫外线过滤器和指定曝光相机和曝光相机2。设置曝光时间为800毫秒1和40毫秒的曝光的曝光2。 </li><li>选择“显微镜”选项卡。定义电子邮件xposure 1 488激光（FITC）和曝光UV（365激光）。激活两次曝光的紫外线过滤器和指定曝光相机和曝光相机2。设置曝光时间为800毫秒1和40毫秒的曝光的曝光2。 </li><li>选择曝光1。将焦点设置为0微米的高度。选择“焦点访谈”。一旦集中，让相机1。调整的重点高度优化曝光平面，点击“采取高度”。改变曝光时间和激光功率〜3,000，得到最大像素强度。保存的曝光参数。重复曝光2。 </li><li>选择“实验定义”选项卡。创建布局和sublayout。拖放相关的布局，曝光，参考图像，skewcrop文件和sublayout。保存实验。 </li><li>选择“自动实验”选项卡，采集图像。 </li></ol> 6。图像分析（基于ImageJ的1.46h版）。 <ol><li>转换的专有珀金埃尔默。Flex文件标记图像格式。tif文件使用Flex2Volocity的Acapella脚本。 </li><li><ol><li>导入ImageJ的图像的图像序列，导致堆栈。 </li><li>转换成2通道hyperstack的堆栈，选择“图像/ Hyperstack /的协议栈Hyperstack菜单项。的片的数目必须被设置为一半进口堆栈。 </li><li>重复使用图像/重复命令：GFP通道的的重复hyperstack复选框打勾，并指定通道：1-1。 ，此后使用复制的堆栈。 </li></ol></li><li>选择过程/对比度增强的0.01％饱和度的像素，使用堆栈柱状图和转换8位Image/Type/8-bit。 </li><li>定义15像素半径本地自适应分割与对比阈值（参数1）●= 30使用伯恩森算法。勾选的白色物体在黑色背景上“复选框，在”图像/调整/自动本地阈值菜单。 </li><li>分割质量控制：对原始图像分割的对象重叠等高线。 </li><li>征象重新提取颗粒分析：测量面积，平均每一种细胞器的总强度。保存结果文件。 </li><li>打开功能在R中的结果文件，并生成一个直方图。 </li></ol> 7。代表性的成果在正常条件下，Grb2的本地化在整个小区。与生长因子受体的刺激后易位到质膜，并随后被内化进入核内体内，如在图4中示出。能的结合Grb2的一种细胞表面受体的表达足以诱导这种易位。在一个典型的筛选试验中，没有观察到在GFP-Grb2的本地化的改变。然而，当一种蛋白质表达新兵Grb2的一个子蜂窝站点，有在本地化的改变，通过荧光显微镜观察，可以很容易地可视化。例如，表皮生长因子受体表达的GFP-Grb2的内体样结构的重新定位（ 图4）。因此，当施加了全基因组库，它可以预期新型Grb2的结合细胞表面的蛋白质可以被识别。 的细胞表面蛋白的检测并不限于此方法。比如，Dynamin2诱导GFP-Grb2的显示内涵体状招聘（ 图5）中的亚细胞定位的变化。 dynamin2 Grb2的SH3结构域的C-末端结合，并参与的内吞作用的这种复杂的9,10。因此，这种方法使一般蛋白结合复合物的识别，并不仅限于识别SH2结构域的相互作用伙伴。 几种不同类型的易位如本地化到质膜，可以预期，内涵体，以及其他胞质泡状结构或细胞核。因此，建议所有图像都还考察了眼睛。然而，当大集的筛选cDNA的一个自动化更实用的图像分析算法。在这种情况下，组合的算法是可取的，如现场检测，以确定内涵体，细胞质/细胞核的检测，以确定核穿梭和一般的形态算法，以确定蜂窝形状的变化。使用这些不同的表型检测方法已别处11更详细地讨论。 <img alt="图1" src="/files/ftp_upload/4382/4382fig1.jpg" /> 图1。总体方案的实验。实验细节开始的放大制备的cDNA文库，转基因向量和记者的结构，图像采集和图像分析使用的是免费开放的软件ImageJ的一个完整的高内涵筛选的工作流程。 <img alt="图2" src="/files/ftp_upload/4382/4382fig2.jpg" /> FIGURE 2。配置的Tecan公司甲板。 （1）在左手侧，5个100毫升的槽需要被填充缓冲区1（40毫升），2（40毫升），3（50毫升），AW（60毫升），A4（100毫升）。海槽A4将需要重新填充在手术过程中一次。 （2）的真空歧管位于在这个例子中的位置14上。 （3）与细菌的颗粒的深孔板位于位置30上的，用25毫升的洗脱缓冲液洗脱缓冲液槽旁边。 （4）8通道头的一次性提示需要加载在头架的左侧和技巧需要填写的40位多声道头。在这个实验中的的Tecan公司FreedomEvo液体处理程序，用于配备了500微升注射器。 <a href="http://www.jove.com/files/ftp_upload/4382/4382fig2large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/4382/4382fig3.jpg" /> 图3。在Opera LX的图像采集的自动化。 A）选择“配置”选项卡（1）。激活的实验（2）所需的激光线。选择的摄像机，用于图像采集（3）。选择板型和物镜，用于试验（4）。 B）选择在显微镜片（1）。定义的光源和用于曝光1（2,3）的曝光时间。专注于一井和高度（4）。 C）选择的实验定义“选项卡（1）。拖放曝光，skewcrop，参考，的布局和sublayout文件（2）。拖放实验文件（3）。 D）实验（1）选择“自动”。拖放实验文件（2）。分配适当的条码（3）。图像采集，通过点击开始按钮（4） <a href="http://www.jove.com/files/ftp_upload/4382/4382fig3large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图4" src="/files/ftp_upload/4382/4382fig4.jpg" /> <stroNG&gt;图4。用cDNA表达GFP-Grb2的易位。 GFP-Grb2的重新定位，从一个占主导地位的胞浆内体样结构的跨膜生长因子受体过度表达时，其后内化到内涵体。在左侧，显​​示的COS M6细胞的显微镜图像，瞬时转染用GFP-Grb2的（顶部）或GFP的，Grb2的加表皮生长因子受体（底部）。表达的表皮生长因子受体在搬迁GFP-Grb2的内体样结构的结果。 <img alt="图5" src="/files/ftp_upload/4382/4382fig5.jpg" /> 图5实施例的cDNA-诱导的GFP-Grb2的易位。的GTP酶Dynamin2，这是参与中Grb2的介导的内吞作用，重新定位GFP-Grb2的内体样结构（圆圈内）。在这个实验中，GFP-Grb2的共转染HEK293T细胞DNM2。细胞用Hoechst 33342染色后24 h和图像被收购的OPERA LX。显示绿色（GFP）的信道的图的一个字段和UV（蓝色，Hoechst 33342荧光）。

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	<li>Sastry, L., Cao, T., King, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Grb2-protein+complexes+in+human+cancer+cells.">Multiple Grb2-protein complexes in human cancer cells.</a> Int. J. Cancer. 70, 208-213 (1997).</li><li>Li, S., Couvillon, A. D., Brasher, B. B., Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosine+phosphorylation+of+Grb2+by+Bcr/Abl+and+epidermal+growth+factor+receptor:+a+novel+regulatory+mechanism+for+tyrosine+kinase+signaling.">Tyrosine phosphorylation of Grb2 by Bcr/Abl and epidermal growth factor receptor: a novel regulatory mechanism for tyrosine kinase signaling.</a> Embo. J. 20, 6793-6804 (2001).</li><li>Bisson, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selected+reaction+monitoring+mass+spectrometry+reveals+the+dynamics+of+signaling+through+the+GRB2+adaptor.">Selected reaction monitoring mass spectrometry reveals the dynamics of signaling through the GRB2 adaptor.</a> Nat. Biotechnol. 29, 653-658 (2011).</li><li>Ketteler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Feynman+trajectories:+determining+the+path+of+a+protein+using+fixed-endpoint+assays.">The Feynman trajectories: determining the path of a protein using fixed-endpoint assays.</a> J. Biomol. Screen. 15, 321-326 (2010).</li><li>Yamazaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Grb2+in+EGF-stimulated+EGFR+internalization.">Role of Grb2 in EGF-stimulated EGFR internalization.</a> J. Cell. Sci. 115, 1791-1802 (2002).</li><li>Antczak, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain-Based+Biosensor+Assay+to+Screen+for+Epidermal+Growth+Factor+Receptor+Modulators.">Domain-Based Biosensor Assay to Screen for Epidermal Growth Factor Receptor Modulators.</a> in Live Cells. ASSAY and Drug Development Technologies. 10, 24-36 (2012).</li><li>Su, J., Batzer, A., Sap, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+tyrosine+phosphatase+R-PTP-alpha+is+tyrosine-phosphorylated+and+associated+with+the+adaptor+protein+Grb2.">Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2.</a> J. Biol. Chem. 269, 18731-18734 (1994).</li><li>Hertog, J. d. e. n., Tracy, S., Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+receptor+protein-tyrosine+phosphatase+alpha+on+Tyr789,+a+binding+site+for+the+SH3-SH2-SH3+adaptor+protein+GRB-2+in+vivo.">Phosphorylation of receptor protein-tyrosine phosphatase alpha on Tyr789, a binding site for the SH3-SH2-SH3 adaptor protein GRB-2 in vivo.</a> Embo. J. 13, 3020-3032 (1994).</li><li>Miki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+Ash/Grb-2+with+dynamin+through+the+Src+homology+3+domain.">Association of Ash/Grb-2 with dynamin through the Src homology 3 domain.</a> J. Biol. Chem. 269, 5489-5492 (1994).</li><li>Seedorf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamin+binds+to+SH3+domains+of+phospholipase+C+gamma+and+GRB-2.">Dynamin binds to SH3 domains of phospholipase C gamma and GRB-2.</a> J. Biol. Chem. 269, 16009-16014 (1994).</li><li>Carpenter, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: image analysis software for identifying and quantifying cell phenotypes.</a> Genome Biol. 7, R100 (2006).</li><li>Seed, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+in+expression+cloning.">Developments in expression cloning.</a> Curr. Opin. Biotechnol. 6, 567-573 (1995).</li></ol>

没有利益冲突的声明。

表达克隆是一种强大的工具，已经被用于在过去，如病毒受体和血细胞抗原12，以确定新的细胞成分。在这里，我们描述了一种方法，以方便识别新的假定的信号转导受体结合GRB2。 在协议中，有几个关键步骤。 <ol><li>对于自动质粒制备，它是绝对必要的，有完整的细菌沉淀悬浮。有时，它是必要的，以包括严格的震动步骤或进一步混合移液器的。 </li><li>待加入溶液2和3，完成混合是必不可少的，并且我们已经发现，手动反相的密封板是更有效比一个自动化摇床使用。移液应避免在这一步，因为它会导致的DNA剪切。 </li><li>在所有的真空步骤，以确保液体已完全耗尽从板。否则，低收益率是可以预期的。 </li><li>洗脱后，这是很常见的，洗脱液滴形式的质粒结合板的底部。这些滴需要收集的洗脱板中，所以它被建议质粒结合板抬起仔细和任何残留的滴在底部小心转入到的洗脱液板。 </li><li>在转染过程中，复合物的形成的时间是至关重要的。我们通常坚持20-30分钟。更长的时间可长达1小时的罚款不减产，不推荐使用，而更短的时间。 </li><li>对于显微镜的倍率的选择是重要的。在Opera LX系统用在这里，是足以获得高品质的图像分辨率为20倍。如果要求更高分辨率的图像，目的是可以改变的，但为了得到统计上显着数量的细胞，较高数目的字段和更高的采集时间是必需的。在一般情况下，我们的目标是得到至少100个细胞，每孔成像。 </li></ol> Grb2的易位检测已被用于其他组的小分子抑制剂的表皮生长因子受体激酶的活化6。在这种情况下，具体激活的表皮生长因子受体与配体引起招募Grb2的质膜。破坏它们之间的相互作用，然后，可以使用小分子化合物的研究。同样，可以设想，可以采用的siRNA屏幕识别参与EGFR-Grb2的信令或其他Grb2的结合如c-KIT或促红细胞生成素受体的细胞生长因子受体的内源性基因。因此，存在多个潜在的应用这种技术。一个类似的方法可以适用于GFP标记的报告系统为其他适配器分子如SHC，加布或IRS。 使用这种基于细胞的测定，在哺乳动物细胞中的一个主要优点是，它能够识别的生理相对evant的相互作用。该检测方法是读出的蛋白复合物的形成，但更重要的是，有关的相互作用是在正确的子蜂窝站点监视。在这方面，这种技术克服工件如酵母双杂交，或在体外测定方法从其他的蛋白-蛋白相互作用的。应该指出，间接的相互作用也可能导致Grb2的招聘。类似的，具有约束力的合作伙伴的转录调控作用可能是通过cDNA表达。为了区分这些可能性，它是需要进行适当的辅助检测区分直接和间接结合效果。 总之，GFP适配器的分子易位试验的承诺全基因组的筛选和药物发现应用潜力。

<table border="1"><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> cDNA文库</td><td> Origene </td><td></td><td></td></tr><tr><td> LB +放大器</td><td></td><td></td><td></td></tr><tr><td>透气性的密封件</td><td> ThermoScientific </td><td> AB-0718 </td><td></td></tr><tr><td> Nucleospin 96质粒试剂盒</td><td> MACHEREY-NAGEL </td><td> 740625.4 </td><td></td></tr><tr><td> Transfectin </td><td> BioRad公司</td><td> 170-3351 </td><td></td></tr><tr><td>形态学</td><td>分子探针</td><td> H3570 </td><td></td></tr><tr><td> Viewplate 96楼TC </td><td>珀金埃尔默</td><td> 6005182 </td><td></td></tr><tr><td> RapidPick </td><td>哈得逊</td><td></td><td>诺冠CP7200 </td></tr><tr><td>自由埃沃</td><td> Tecan公司</td><td></td><td>随着真空歧管</td></tr><tr><td>多点384 </td><td>热</td><td></td><td></td></tr><tr><td>歌剧LX </td><td>珀金埃尔默</td><td></td><td></td></tr></table>

a high-content imaging workflow to study grb2 signaling complexes by expression cloning

生长因子受体信号转导的维持细胞的增殖和分化是必要的，需要严格控制。信号转导是由的外部配体结合到跨膜受体，激活下游的信号通路。一个关键的调节有丝分裂信号是Grb2的，模块化的内部SH2（Src同源2）域两侧的两个SH3结构域，缺乏酶的活性的蛋白质组成。 Grb2的是组成与GTP酶的儿子作者-Sevenless（SOS）相关联，通过其N-末端SH3结构域。 SH2结构域的Grb2的结合生长因子受体磷酸化酪氨酸残基偶联受体激活的SOS-RAS-MAP激酶信号级联。此外，作为正的或负的稳压器的信令和受体的内吞作用的Grb2的其他角色已被描述。 Grb2的模块化的组合物，表明，它可以停靠各种受体和转导E信号沿许多不同的途径1-3。 这里描述的是一个简单的显微镜检测，监控招募到质膜的Grb2的。它适于从一个试验，其测量变化的亚细胞定位的绿色荧光蛋白（GFP）的标签Grb2的响应于刺激4-6。质膜受体结合GRB2如激活表皮生长因子受体（EGFR）招GFP-Grb2的cDNA表达的质膜后，随后搬迁到在细胞中的胞内舱。为了识别体内 Grb2的蛋白复合物的，这种技术可以用于执行了全基因组基于高含量屏幕的变化Grb2的亚细胞定位。在下面详细描述的制备的cDNA的表达克隆，转染和图像采集。用于识别蛋白质相互作用伙伴的其他基因组的方法，如酵母双-HY相比杂种，这种技术允许在哺乳动物细胞中的蛋白复合物的可视化，在通过一个简单的显微镜为基础的检测的相互作用的亚细胞站点。因此，定性特征，诸如图案的本地化可评估，以及作为定量的相互作用强度。

Watch this Scientific Journal Video about A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning at JoVE.com

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

高内涵筛选新的信号主管跨膜受体的鉴定方法。此方法是适合于大规模的自动化，并允许的预测<em在体内</em&gt;蛋白结合和在哺乳动物细胞中的蛋白复合物的亚细胞定位。

一个高含量的成像流程研究Grb2的信号复合体的表达克隆

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

a-high-content-imaging-workflow-to-study-grb2-signaling-complexes

Research

JoVE Journal

Biology

10.5K 次观看.  University College London. 高内涵筛选新的信号主管跨膜受体的鉴定方法。此方法是适合于大规模的自动化，并允许的预测

视频: A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

依赖的PDZ-CFTR的大分子信号复合物的体外分析

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代谢率的测量<em&gt;果蝇</em&gt;使用呼吸运动

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators.
Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software1&#160;to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.

工作流的高含量，荧光标记的万能工具显微镜数据，支持开源软件的单个细胞定量

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly ...

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 ...