作者会喜欢到感谢博士斯蒂芬金斯莫尔和的儿科基因组医学中心儿童慈悲医院和诊所为他们的计算集群为我们的数据分析的，Illumina公司的现场服务团队（伊丽莎白·博耶，斯科特·库克和马克库克）和技术顾问团队，为他们的快速反应和下一代DNA测序仪，HiScanSQ，和数据质量分析的运行有用的建议。这项工作是支持的，部分由美国国立卫生格兰特HL080042（SQY）启动基金和养老的儿童慈善医院和诊所在堪萨斯城，密苏里大学（SQY）。

<html>概述本协议的流程图显示在图1。 1。处理过的细胞凝血酶，总RNA的提取，质量评价和定量RNA <ol><li>培养人肺微血管内皮细胞（HMVEC-LBL）90-100％汇合在EGM-2培养基的6孔板中，5％FBS，生长因子和抗生素（龙沙，猫＃CC-3202）。 </li><li>变更媒体饥饿媒体（0％FBS）30分钟前，用凝血酶处理。 </li><li>治疗的细胞用0.05 U / ml凝血酶或留下未处理的作为控制6小时，在37℃和5％CO 2。 </li><li>根据制造商的说明使用Ambion公司的的MIR瓦纳套件组和对照组的细胞，提取总RNA。 </li><li>根据标准协议，在Experion的全自动电泳站（评估质量的RNA与的Experion StdSense真核RNA芯片的="_blank"&gt;生物技术有限公司www.bio-rad.com）。 </li><li>量化的RNA，使用标准的分光光度测定方法。 </li></ol> 2。图书馆的建设和测序<ol><li>使用高品质的每个样品的总RNA作为起始原料的1微克。 </li><li>要构建库，按照标准的程序，从Illumina公司（协议＃15008136修订版A）。在这个协议中，两回合的poly（A）含有基因的选择进行删除rRNA基因，以尽量减少rRNA序列。 </li><li>评估质量的图书馆使用的Experion DNA 1K芯片根据协议在Experion自动电泳站（ <a href="http://www.bio-rad.com" target="_blank">生物技术有限公司www.bio-rad.com</a> ）的标准。 </li><li>使用定量PCR量化库:使用一个库，先前已被测序的标准曲线和特异性引物的连接适配器。未知的库（ 即 1:100，1:500和1:1000）稀释的使用范围。运行的qPCR ACCO富于到的SyberGreen的MM协议，并计算每个库的原始股票的浓度。 </li><li> 10纳米和储存在-20°C，直到准备群集流动池稀释库股票。 </li><li>在准备群集流动池，在水浴中解冻CBOT试剂板。 CBOT是一个Illumina公司的仪器，用于简化集群生成过程。 </li><li>洗芝加哥期货交易所的仪器。 </li><li>变性图书馆:联合13微升1×TE和6μl10μM的库，侧管，加入1微升的1 N NaOH（Illumina公司提供）。漩涡，旋转，室温孵育5分钟，放置在冰的变性库。 </li><li>稀释库:用预先冷却的杂交缓冲液中稀释的变性图书馆（HT1，Illumina公司提供）由结合下午12点的最终浓度为996微升HT1和4微升变性库。将冰的变性及摊薄库。 </li><li>反转每一行的CBOT板，管，确保所有试剂融化。自旋向下的板块，删除/穿刺的铝箔密封件，并加载到芝加哥期货交易所。 </li><li>等分的120微升稀释的，变性的图书馆的带状管，标记为1-8。每管加入1.2μl稀释，变性PhiX控制库（Illumina公司）有穗的控制。涡街流量计和旋管，并加载它们在正确的方向芝加哥期货交易所（管＃1在右边）。 </li><li>到芝加哥期货交易所加载的流动池和歧管。 </li><li>完成流量检查，并开始运行的集群。 </li><li>运行完成后，检查试剂提供的所有车道。请注意任何异常。无论是立即开始测序运行流动池或存储在所提供的管，在4℃下</li><li>解冻合成边测序（SBS，Illumina公司）试剂。 </li><li>装入试剂的试剂盘到适当的位置，确保不要接触到其他试剂接触后的切割组合。 </li><li>使用无N-测序流通池（ 即一个被测序），两次总理的试剂管线。 </li><li>彻底清洁序流通池，用70％的乙醇和Kimwipes，其次是70％的乙醇和镜头纸。检查任何条纹的流动池。重新必要时进行清理。 </li><li>加载到定序器的流动池，执行流量检查，以确保歧管之间的密封和流动池是紧。 </li><li>启动的顺序运行。 </li><li>评估，因为他们成为可用在运行过程中的质量指标（ 如群密度，通过过滤器的集群，Q30，强度）。 </li><li>监视整个运行强度。 </li><li> 101周期完成后，执行完成第二次读取周转化学:解冻的的配对末端试剂和第二个读法团缓冲器（ICB，SBS试剂的一个组成部分，Illumina公司）和加载的试剂。 </li><li>继续测序运行，评估第二次读强度，Q30一在运行过程中的第二等质量指标。 </li></ol> 3。数据分析<ol><li>使用最新版本的CASAVA（Illumina公司，目前为1.8.2）转换基地调用文件（BCL）文件。fastq文件，fastq簇数为0，以确保每个样品的一个单一的fastq文件的创建。用于下游分析解压缩的fastq的文件。 </li><li>执行成对的末端对齐使用最新版本的顶帽（1.4.1）5，对齐RNA-seq的读取大小的哺乳动物基因组的超高通量短读定位器（蝴蝶结，0.12.7）6和SAMtools（0.1。 17）7。 SAMtools实现各种工具，用于处理后的SAM格式的结盟。参照人类转录组，可以下载从iGenomes（ <a href="http://www.illumina.com" target="_blank">www.illumina.com</a> ）。在运行的顶帽，我们使用默认的参数设置，包括库类型"选项（默认）FR-unstranded。 </li><li>我们8软件包ING方案CuffDiff的，部分的袖扣（1.3.0），比较凝血酶处理的细胞的控制细胞，筛选出的差异表达的基因转录在前者基于人类基准转录。这种比较检测到的差异表达已知的成绩单。使用Microsoft Excel的可视化结果以表格的形式。在运行袖扣程序，我们使用默认参数设置。这些基因的转录与FPKM &lt;0.05和P&gt; 0.05被过滤掉。 </li><li>检测新亚型，运行袖扣无参考转录。比较样本抄本文件的参考基因组Cuffcompare，用合成凝血酶抄本文件作为一个分析和综合控制的参考基因组转录文件作为参考基因组的第二个分析测试的差异表达Cuffdiff，。使用Microsoft Excel在表格的形式来显示结果。同样，茨艾伦E基因转录与FPKM &lt;0.05和P&gt; 0.05被过滤掉。在这一步之后，研究人员可以选择上传一个新报告的成绩单列表的UCSC基因组浏览器网页（ <a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a> ），通过手动检查以验证其有效性。 </li><li>提交列表中差异表达的基因，以别出心裁的途径分析（IPA， <a href="www.ingenuity.com" target="_blank">www.ingenuity.com</a> ）为特征的凝血酶治疗的基因和通路的影响。在这个步骤中，调查人员可以选择使用腰带（ <a href="http://compbio.mit.edu/cummeRbund/" target="_blank">http://compbio.mit.edu/cummeRbund/</a> ）的R套件，旨在帮助和简化的任务分析袖扣RNA-seq的输出，来帮助管理，可视化和整合生产的Cuffdiff分析所有的数据。 </li></ol> 4。验证RNA-seq的结果实时定量宝lymerase链反应（定量RT-PCR） <ol><li>执行提取总RNA控制的凝血酶治疗的HMVEC的-LBL细胞中，RNA质量评估和RNA定量描述的步骤1.4至1.6。 </li><li>产生互补DNA来自1微克的总RNA，每个样品的SuperScript III第一链合成系统的RT试剂盒，按照制造商的说明（Invitrogen公司制，18080-051）。 </li><li>一个美国应用生物系统公司VIIA 7实时PCR系统，利用TaqMan分析的需求设计的寡核苷酸检测CUGBP，ELAV likefamilymember1（CELF1，Hs00198069_m1），Fanconianemia，complementationgroupD2（FANDCD2，Hs00276992_m1），TNFreceptor进行定量RT-PCR分析相关因子1（TRAF1，Hs01090170_m1），和β-肌动蛋白（ACTB，Hs99999903_m1）。每个样品有一个相当于5 ng总RNA的模板。测量定量使用DDCT方法和规范，以β-肌动蛋白。每个试验进行过至少3次生物学重复。 </li></ol>

<ol>
	<li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nat. Rev. Genet. 10, 57-63 (2009).</li><li>Shendure, J., Ji, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+DNA+sequencing.">Next-generation DNA sequencing.</a> Nat. Biotechnol. 26, 1135-1145 (2008).</li><li>Marioni, J. C., Mason, C. E., Mane, S. M., Stephens, M., Gilad, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-seq:+an+assessment+of+technical+reproducibility+and+comparison+with+gene+expression+arrays.">RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays.</a> Genome Res. 18, 1509-1517 (2008).</li><li>Zhang, L. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-seq+Reveals+Novel+Transcriptome+of+Genes+and+Their+Isoforms+in+Human+Pulmonary+Microvascular+Endothelial+Cells+Treated+with+Thrombin.">RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin.</a> PLoS One. 7, e31229 (2012).</li><li>Trapnell, C., Pachter, L., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopHat:+discovering+splice+junctions+with+RNA-Seq.">TopHat: discovering splice junctions with RNA-Seq.</a> Bioinformatics. 25, 1105-1111 (2009).</li><li>Langmead, B., Trapnell, C., Pop, M., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+and+memory-efficient+alignment+of+short+DNA+sequences+to+the+human+genome.">Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.</a> Genome Biol. 10, R25 (2009).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sequence+Alignment/Map+format+and+SAMtools.">The Sequence Alignment/Map format and SAMtools.</a> Bioinformatics. 25, 2078-2079 (2009).</li><li>Trapnell, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcript+assembly+and+quantification+by+RNA-Seq+reveals+unannotated+transcripts+and+isoform+switching+during+cell+differentiation.">Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation.</a> Nat. Biotechnol. 28, 511-515 (2010).</li><li>Khatri, P., Sirota, M., Butte, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ten+years+of+pathway+analysis:+current+approaches+and+outstanding+challenges.">Ten years of pathway analysis: current approaches and outstanding challenges.</a> PLoS Comput. Biol. 8, e1002375 (2012).</li><li>Nielsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+with+RNA.">Working with RNA.</a> Methods. Mol. Biol. 703, 15-28 (2011).</li><li>Trapnell, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+gene+and+transcript+expression+analysis+of+RNA-seq+experiments+with+TopHat+and+Cufflinks.">Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.</a> Nat. Protoc. 7, 562-578 (2012).</li><li>Robertson, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+assembly+and+analysis+of+RNA-seq+data.">De novo assembly and analysis of RNA-seq data.</a> Nat. Methods. 7, 909-912 (2010).</li><li>Garber, M., Grabherr, M. G., Guttman, M., Trapnell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+methods+for+transcriptome+annotation+and+quantification+using+RNA-seq.">Computational methods for transcriptome annotation and quantification using RNA-seq.</a> Nat. Methods. 8, 469-477 (2011).</li><li>Martin, J. A., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+transcriptome+assembly.">Next-generation transcriptome assembly.</a> Nat. Rev. Genet. 12, 671-682 (2011).</li></ol>

没有利益冲突的声明。

关键步骤 RNA处理核糖核酸酶会降低，即使是最优质的RNA，因此，护理时必须采取相应的隔离，储存和使用的RNA 10。总是戴手套，以防止污染由核糖核酸酶中发现人的手。手套应经常更换，尤其是接触皮肤后，门把手或其他常见的表面。一组吸液管应专门的RNA工作，所有的技巧和管应无RNA酶。 RNA分离和下游的应用程序，应进行低流量区域，常规的RNase-扎普如...

<table border="1"><tbody><tr><td width="183"> 试剂和设备 </td><td width="85"> 公司 </td><td width="185"> 目录号 </td><td width="359"> 评论 </td></tr><tr><td>人肺微血管内皮细胞</td><td>龙沙</td><td> CC-2815 </td><td></td></tr><tr><td>龙沙，子弹套件</td><td>龙沙</td><td> CC-3202 </td><td>包含EGM-2，FBS，生长因子和抗生素</td></tr><tr><td>凝血酶</td><td>西格玛</td><td> T4393 </td><td></td></tr><tr><td> Ambion公司MIR瓦纳套件</td><td> Life Technologies公司</td><td> AM 1560 </td><td></td></tr><tr><td>核糖核酸酶-ZAP </td><td> Life Technologies公司</td><td> AM9782 </td><td></td></tr><tr><td> Experion的StdSens RNA </td><td> Bio-Rad公司</td><td> 700-7103 </td><td></td></tr><tr><td> TruSeq RNA的制备试剂盒</td><td> Illumina公司</td><td> FC-122-1001 </td><td></td></tr><tr><td> AMPureXP珠</td><td> Beckman Coulter公司</td><td> A63881 </td><td></td></tr><tr><td>标逆转录酶II </td><td> Life Technologies公司</td><td> 18064-014 </td><td></td></tr><tr><td> Experion的DNA 1K </td><td> Bio-Rad公司</td><td> 700-7107 </td><td></td></tr><tr><td> QuantiTect SyberGreen </td><td> Qiagen公司</td><td> 204163 </td><td></td></tr><tr><td> PE集群生成工具包</td><td> Illumina公司</td><td> PE-401-3001 </td><td></td></tr><tr><td> PhiX控制工具包</td><td> Illumina公司</td><td> FC110-301 </td><td></td></tr><tr><td> 200周期SBS套件</td><td> Illumina公司</td><td> FC-401-3001 </td><td></td></tr><tr><td> HiScanSQ * </td><td> Illumina公司</td><td> SY-103-2001 </td><td></td></tr><tr><td>芝加哥期货交易所</td><td> Illumina公司</td><td> SY-301-2002 </td><td></td></tr><tr><td>定量PCR机 - Viia7台</td><td> Life Technologies公司</td><td>型号＃VIIA7 /设备＃10631261 </td><td>或同等学历</td></tr><tr><td> Experion系统</td><td> BIO RAD </td><td> 7007001 </td><td>生物分析仪是一种替代系统</td></tr><tr><td>分光光度计</td><td> Bio-Tek的</td><td>大纪元微孔板分光光度计</td><td>或同等学历</td></tr><tr><td>离心机 - SORVALL传奇XTR </td><td> Thermo Scientific的</td><td> 75004521 </td><td>或同等学历</td></tr><tr><td>磁性底座</td><td> Life Technologies公司</td><td> AM10027 </td><td></td></tr><tr><td> 96孔热循环仪</td><td>一般实验室供应商</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> 表3列出主要试剂和主要电子quipment。 *，在视频中，HiSeq1000证明而不是HiScanSQ的。 </td></tr></tbody></table>

rna-seq analysis of transcriptomes in thrombin-treated and control human pulmonary microvascular endothelial cells

通过测量细胞中的mRNA水平的基因表达的特征是确定如何转录机制的细 ​​胞受到外部信号（ 如药物治疗），或不同的细胞如何健康状态和疾病状态之间的一个有用的工具。随着新一代DNA测序技术的出现和不断改进，RNA测序（RNA-seq的）已成为越来越受欢迎的转录组分析的方法进行编目所有种类的成绩单，以确定所有基因表达的转录结构和量化的表达水平的变化在一个给定的细胞，组织或生物体1,2的总组转录。 RNA-Seq是逐渐取代DNA微阵列转录组分析的首选方法，因为它有一个完整的转录组分析的优势，提供一个数字型数据（拷贝数的任何誊本），而不是依靠任何已知的基因组sequenCE 3。 在这里，我们提出了一个完整的，详细的协议，适用个人资料转录RNA-seq的人肺微血管内皮细胞或无凝血酶治疗。该协议是基于我们最近发表的研究报告，题为"RNA-seq的揭示新型转录的基因及其亚型在人肺微血管内皮细胞凝血酶治疗，"4中，我们成功地完成了第一个完整的人肺微血管内皮细胞的转录组分析与凝血酶使用RNA-seq的治疗。进一步的实验，取得了前所未有的资源，获得洞察的分子机制，凝血酶介导的内皮功能紊乱的炎症性疾病，癌症，糖尿病和冠状动脉心脏疾病的发病机制，并为这些疾病的治疗目标，以提供潜在的新的线索。 此协议的描述性文本被分成四个部分。第一部分介绍了人肺微血管内皮细胞的凝血酶和RNA的提取，质量分析和定量分析的治疗。第二部分介绍了图书馆的建设和测序。第三部分描述了数据分析。第四部分描述的RT-PCR验证试验。显示了有代表性的几个关键步骤的结果。在讨论中提供有用的提示或预防措施，以提高成功的关键步骤。虽然该协议使用人肺微血管内皮细胞用凝血酶，它可以是广义在哺乳动物和非哺乳动物的细胞和组织中具有不同刺激物或抑制剂，或在细胞或组织之间的健康状态来比较转录处理的档案中转录和疾病状态。

对于步骤1:28秒:18比传统使用的RNA的降解是一个指标。在理想的情况下，28秒峰值应具有约两倍的面积的18带（2）的比率，但是这理想比例往往不能在实践中看到。此外，28秒:18秒从分光光度测定方法得到的比率可以低估的量的RNA的降解。为了更精确地量化的退化，并因此的RNA样品的质量，的Experion系统计算的RNA质量指示符（RQI）号码。 RQI算法RNA样品的电泳图谱进行比较，从一系列的?...

Watch this Scientific Journal Video about RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells at JoVE.com

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

该协议提供了一个完整而详细的程序，申请一个功能强大的下一代DNA测序技术，RNA-seq技术，在人肺微血管内皮细胞或无凝血酶治疗的个人资料转录。这个协议是一般化到各种细胞或组织中的影响不同的试剂或疾病状态。

RNA-seq的凝血酶处理和控制人肺微血管内皮细胞的转录组分析

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

rna-seq-analysis-transcriptomes-thrombin-treated-control-human

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

21.8K 次观看.  Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City. 该协议提供了一个完整而详细的程序，申请一个功能强大的下一代DNA测序技术，RNA-seq技术，在人肺微血管内皮细胞或无凝血酶治疗的个人资料转录。这个协议是一般化到各种细胞或组织中的影响不同的试剂或疾病状态。

视频: RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

分离人脐静脉内皮细胞（HUVEC）

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

科研

生物学

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

生物人类免疫缺陷小鼠的微血管网络

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

人鼻粘膜上皮细胞在气液界面培养

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

从鼠标阻力动脉分离微血管内皮管

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

一氧化氮和超氧化物歧化酶恩的人脸检测在完整的动脉内皮细胞层

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.