这项研究是支持的资金丽塔艾伦基金会授予Seena阿吉特。笔者想从南卡罗来纳州电子显微镜仪器的使用，科学和技术援助中心大学的埃里卡巴洛格和Soumitra Ghoshroy博士的承认。

所有实验中使用人类和啮齿动物的血液样本，符合所有有关准则，法规和监管机构执行。人类受试者参加知情同意后批准的德雷克塞尔大学Drexel的机构动物护理和使用委员会批准使用动物进行研究医学伦理审查委员会的一切手续。 1。外切体净化血液（约5.5小时） TIPS <ul><li class="jove_content">我们重悬外来一管血300μlRNA提取裂解液从mirVana miRNA分离试剂盒（〜10毫升人体血液和小鼠血〜2毫升）。 </li><li class="jove_content">为了纯化从细胞培养基的外来体，介质旋转，在500×g离心10分钟以除去细胞，并以12,000×g离心分离后，通过0.22微米的过滤器过滤。外切体净化细胞培养基不需要稀释在PBS，PBS洗或的第二超速离心步骤。 </li><li class="jove_content">如果纯化的外来体将用于蛋白质组学研究，超速离心步骤后，加入蔗糖梯度离心步骤将减少量的蛋白聚集体和污染物15。 </li><li class="jove_content">外来体可以观察到的形态有所不同，这取决于同时在源和用于TEM的处理步骤。我们并没有观察到样品处理9,15差异归因于先前已外来杯形形态。我们的TEM照片，包括免疫标记类似于以前的报告从不同的来源，其中包括血液，胆汁和皮质文化衍生的外来9,16,17。 </li></ul><ol><li>反转涂覆EDTA管中，血液5倍和地点直立10〜60分钟，在室温下。 </li><li>在4℃下2000×g离心15分钟离心</LI&gt; <li>收集的顶层，血浆，在15毫升管中并保持在冰上。 </li><li>稀释液用等体积的PBS。以2000×g离心30分钟，在4℃下</li><li>转入离心管，加的总体积为24ml的PBS洗涤，并在4℃下以12000×g离心45分钟</li><li>转移到超速离心机管，并以110,000 xg离心2小时，在4°C </li><li>重悬沉淀在PBS中，在4℃下以100,000 xg离心1小时</li><li>在适当的缓冲液中悬浮颗粒（RNA裂解缓冲液）。 </li><li>重悬在PBS或RIPA缓冲电子显微镜和免疫印迹分析。 </li></ol> 2。 RNA纯化用改进的mirVana miRNA分离试剂盒（约1小时） TIPS <ul><li class="jove_content">佩戴个人防护装备（PPE），分离RNA核糖核酸ZAP擦拭表面和移液器，并使用无菌过滤嘴和管准备的区域。 </li><li类=的“jove_content”&gt;从供应商的协议虽然给出了一个选项净化唯一的miRNA，我们总是提取总RNA，包括基因和miRNA，这样我们就可以研究无论是从同一样品。 </li><li class="jove_content">添加一个列上的DNase处理步骤，除去可能存在的DNA痕迹。根据外来的来源，DNA已经从一些消息来源，包括鼠标和人体的肥大细胞线8，但已发现染色体的DNA序列外来媒体纯化心肌细胞的18纯化外来缺席。 </li><li class="jove_content">我们减少洗涤液的体积1至350微升（下面的步骤2.4），因为供应商提供足够的清洗液1 700μL此外。如果不降低音量没有足够的解决方案，使用整个套件内容留下。其余的步骤，根据制造商的建议进行。 </li></ul><ol><li>加入1/10 VOmiRNA的的匀浆添加剂（30微升）和孵育10分钟冰lume明。 </li><li>提取物的体积为基磺酸 - 苯酚：氯仿等于初始的溶胞产物的体积。在新的管中，离心回收上层相。 </li><li>添加1.25卷100％的乙醇（375微升）。涡街流量计。适用于过滤盒和以10,000×g离心15秒。 </li><li>加入推荐量的miRNA洗涤液1（350微升）的一半，并以10,000×g离心15秒。 </li><li>对每个样品中加入10微升DNA酶1〜70微升缓冲液RDD（Qiagen公司）。加入80微升列，并在室温下孵育15分钟。 </li><li>添加洗涤液1（350微升），并以10,000×g离心15秒。 </li><li>添加清洗液2/3（500微升），并在10,000×g离心15秒。重复。 </li><li>洗脱用40微升95℃下无核酸酶的H 2 O的</li><li>确定RNA浓度的RNA稀释至100毫微克/微升。 </li></ol> 3。布卢外来体miRNA表达谱d使用Taqman探针低密度阵列（TLDA）的卡（约6.5小时） TIPS <ul><li class="jove_content">通过佩戴个人防护装备，分离RNA核糖核酸ZAP擦拭表面和移液器，并使用无菌过滤嘴和管准备的区域。 </li><li class="jove_content">预扩增步骤是建议（前置放大器）1-350纳克RNA（350-1,000纳克样品不需要前置放大器的一步合成cDNA后）。重要的是要确定是否前置放大器为您的样品是必要的，因为你必须把所有的样品在同等条件下。前置放大器的步骤增加了成本和Ct值有一个较低的可接受的切断（32，而不是40）。 </li><li class="jove_content">它是有用的，而不是根据卡的数量，可以运行在一个单一的天的板，以制备的cDNA的反应在条形管。这将减少在同一天，将不被处理的cDNA样品冻融。 </li><li class="jove_content">外来体从不同来源收集有不同租金额的RNA。此协议允许最多3微升RNA为模板的cDNA合成反应的。根据不同来源，可以被孤立的exosomal RNA的量可能有限。而一个典型的组织会产生足够的RNA的cDNA，将100毫微克的RNA在3微升的体积，从人血或人类细胞培养基的外来通常含有较少的RNA和cDNA的必须由较少的起始原料。小鼠血液中的外来体含有较高水平的RNA，可以通过以下方式获得至少100毫微克3微升。 </li></ul><ol><li>要使用的Megaplex池纯化RNA制备cDNA逆转录引物（甲组及乙组B包含miRNA的引物微阵列卡A和B），冰解冻反应组分。 </li><li>标签两个RNase-free的1.5毫升管RT反应池的引物或乙组的引物，并添加组件图表顺序。添加至少一个额外的元件体积为2个或更多的反应，或使主结构建议采用t他的供应商。 </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> RT反应成分 </td><td> 成分浓度 </td><td> 1个样本的体积（的总V /样品= 4.5微升） </td></tr><tr><td>无核酸酶水</td><td></td><td> 0.20微升</td></tr><tr><td>的Megaplex RT缓冲液（10X） </td><td> 1X </td><td> 0.80微升</td></tr><tr><td> dNTPs浓度（100毫米） </td><td> 4.4毫米</td><td> 0.20微升</td></tr><tr><td> 氯化镁 （25毫米） </td><td> 5毫米</td><td> 0.90微升</td></tr><tr><td> RNase抑制剂（20U/μl） </td><td> 2 U </td><td> 0.10微升</td></tr><tr><td> A或B的Megaplex RT引物（10X） </td><td> 1X </td><td> 0.80微升</td></tr><tr><td> MultiScribe逆转录酶</td><td> 75ü </td><td> 1.50微升</td></tr></tbody></table><ol start="3"><li>反转管6次，占简要rifuge。然后，移液管4.5微升RT反应混合到微安8管条或96孔微安的光学反应板。 </li><li>添加100 ng的RNA和音量调整到3微升DEPC处理过的水或加3μl总RNA，搅拌枪头，密封管或板。短暂离心，并在冰上孵育5分钟。 </li><li>将反应到PCR仪，并在下列条件下运行（建议由美国应用生物系统公司的Megaplex池协议）： </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> 舞台 </td><td> 温度 </td><td> 时间 </td></tr><tr><td>周期（40X） </td><td> 16°C </td><td> 2分钟</td></tr><tr><td></td><td> 42°C </td><td> 1分钟</td></tr><tr><td></td><td> 50°C </td><td> 1秒</td></tr><tr><td>持有</td><td> 85°C </td><td> 5分钟</td></tr><tr><td>持有</td><td> 4°C </td><td> ∞ </td></tr></tbody></table><ol start="6"><li>储存在-20℃下（至少一个星期好）的cDNA或继续预放大器的步骤可以被存储为1个星期。 </li><li>甲组和乙组的冰解冻前置放大器的引物，并转化为混合。漩涡Taqman探针前置放大器主也并保持在冰上。 </li><li>按照图表添加至少一个额外的元件体积为2个或更多的反应，或由供应商建议的高手组合。 </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> 前置放大器反应成分 </td><td> 1个样本的音量 </td></tr><tr><td>无核酸酶水</td><td> 7.5微升</td></tr><tr><td> Taqman探针前置主混音（2X） </td><td> 12.5微升</td></tr><tr><td> A或B的Megaplex前置放大器引物（10X） </td><td> 2.5微升</td></tr></tbody></table><ol start="9"><li>吸取2.5μLRT机生产线克拉到8管带或96孔微安的光学反应板和分配22.5微升前置放大器各孔中的反应混合液到微安。 </li><li>密封板，在冰上孵育5分钟，然后装入样品放入PCR仪。在下列条件下运行。 </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> 舞台 </td><td> 温度 </td><td> 时间 </td></tr><tr><td>持有</td><td> 95°C </td><td> 10分钟</td></tr><tr><td>持有</td><td> 55°C </td><td> 2分钟</td></tr><tr><td>持有</td><td> 72°C </td><td> 2分钟</td></tr><tr><td>周期（12X） </td><td> 95°C </td><td> 15秒</td></tr><tr><td></td><td> 60°C </td><td> 4分钟</td></tr><tr><td>持有</td><td> 99.9°C </td><td> 10分钟</td></tr><tr><td>持有</td><td> 4°C </td><td> ∞ </td></tr></tbody></table><ol start="11"><li>前置放大器反应结束后，短暂离心管或板，然后向每孔或管中加入75μl的0.1X TE pH为8.0。 </li><li>密封和混合简单旋转。稀释后的产品存放在-20℃下为1个星期，或继续加载板。 </li><li>组合下列RNase-free的1.5毫升管中，在冰上。 </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> 元件 </td><td> 1卡的体积 </td></tr><tr><td> TaqMan通用PCR预混无AmpErase UNG（2X） </td><td> 450微升</td></tr><tr><td>摊薄前置放大器产品</td><td> 9微升</td></tr><tr><td>无核酸酶水</td><td> 441微升</td></tr></tbody></table><ol start="14"><li>反转管混合并短暂离心​​。免除100微升PCR反应混合成的TaqMan microRNA的阵列卡的每个端口。 </li><li>离心卡2×1,000×g离心1分钟。密封卡，并运行它使用Applied Biosystems公司手册和384 Taqman探针低密度阵列设置中的引物提供的模板。我们使用使用Applied Biosystems 7900HT快速实时PCR系统。 </li></ol> 4。 mRNA的分析采用TaqMan（约1.5小时） <ol><li>要准备的qPCR反应，反应组分混合，在图表中的顺序。所有的样品进行分析与感兴趣的作为标准的基因和18S rRNA基因的基因的引物探针。 </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Taqman探针反应成分 </td><td> 1个样本的音量 </td></tr><tr><td>无核酸酶水</td><td> 7微升（调整个别样品的基础上） </td></tr><tr><td> Taqman探针快速预混（2X） </td><td> 10微升</td></tr><tr><td> Taqman探针引物探针（20倍） </td><td> 1微升</td></tr><tr><td>从100纳克RNA基因</td><td> 2亩L（调整基于个别样本） </td></tr></tbody></table><ol start="2"><li>运行（使用Applied Biosystems 7900HT快速实时PCR系统）制造商的建议，使用快速的96块96孔板。 </li></ol>

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L., Mathivanan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+proteomic+insights+and+diagnostic+potential.">Exosomes: proteomic insights and diagnostic potential.</a> Expert Rev Proteomics. 6, 267-283 (2009).</li><li>Turchinovich, A., Weiz, L., Langheinz, A., Burwinkel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+extracellular+circulating+microRNA.">Characterization of extracellular circulating microRNA.</a> Nucleic acids research. 39, 7223-7233 (2011).</li><li>El-Andaloussi, S., 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=Exosome-mediated+delivery+of+siRNA+in+vitro+and+in+vivo.">Exosome-mediated delivery of siRNA in vitro and in vivo.</a> Nature. 7, 2112-2126 (2012).</li><li>Singh, P. P., Smith, V. L., Karakousis, P. C., Schorey, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+isolated+from+mycobacteria-infected+mice+or+cultured+macrophages+can+recruit+and+activate+immune+cells+in+vitro+and+in+vivo.">Exosomes isolated from mycobacteria-infected mice or cultured macrophages can recruit and activate immune cells in vitro and in vivo.</a> J. Immunol. 189, 777-785 (2012).</li><li>Etheridge, A., Lee, I., Hood, L., Galas, D., Wang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+microRNA:+A+new+source+of+biomarkers.">Extracellular microRNA: A new source of biomarkers.</a> Mutat. Res. , (2011).</li><li>Stenvang, J., Silahtaroglu, A. N., Lindow, M., Elmen, J., Kauppinen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+LNA+in+microRNA-based+cancer+diagnostics+and+therapeutics.">The utility of LNA in microRNA-based cancer diagnostics and therapeutics.</a> Seminars in cancer biology. 18, 89-102 (2008).</li><li>Mathivanan, S., Fahner, C. J., Reid, G. E., Simpson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ExoCarta+2012:+database+of+exosomal+proteins,+RNA+and+lipids.">ExoCarta 2012: database of exosomal proteins, RNA and lipids.</a> Nucleic acids research. 40, D1241-D1244 (2012).</li></ol>

作者什么都没有透露。

在这个协议中，我们展示了通过差速离心纯化的外来从血和文化媒体的miRNA和mRNA的定量。外来体取决于其原产地有不同的组成部分，并参与了一些生物功能，包括免疫反应，抗原呈递细胞内的沟通，转移RNA和蛋白质9,11,12,19,20。大小和形状是外切体纯度的一个决定性因素，一些论文EM外来的数据表明，这些囊泡的范围可以在大小和形态的基础上从哪个源分泌，以及用于固定的方法和成像...

短期非编码的miRNA调节基因表达靶mRNA结合。种子序列〜7个碱基对的互补性使miRNA的结合到靶mRNA，导致翻译抑制或减少的mRNA的稳定性，这两者都可以导致靶蛋白1的表达减少。在过去十年的研究明确证明的基础性作用的miRNA介导细胞功能。同时也出现了相当大的努力，朝向解剖的miRNA介导的分子变化相关的各种疾病2,3。此外，最近鉴定稳定的miRNA在体液4-6铺平了道路，利用他们作为新型生物标志物，适合于临床诊断。 外来体，小囊泡携带基因，蛋白质，脂类介质，和miRNA通过全身血液circulat的受体细胞是通过体液中miRNA的运输模式之一离子7-14。这将导致在受体细胞的基因表达的调制和蜂窝通信代表了一种新的机制。例如，细胞可以调节分泌和/或参与炎症如白细胞介素-1β（IL1β），肿瘤坏死因子-α（TNFα）的含有生物分子的吸收外来的免疫调节过程，转化生长因子-β5（TGFβ5），并调节这些基因的miRNA 13。由于miRNA表达异常在人体多种疾病的共同特点是，这些分子的发现和验证新的治疗目标2提供了激动人心的新机遇。 循环miRNA是存在的所有体液中，它被称为外来的组成是不同的，根据他们被释放源细胞。因此，他们提供了一个途径来研究细胞的生理状况和细胞改变信号甚至TS压力，包括疾病。研究切体成分的改变，可以提供洞察信号转导，并探讨其潜在效用作为生物标志物或治疗干预航线。 在这里，我们将展示发布的协议的基础上，从多个来源的外来的净化。这些将用于RNA分离qPCR的识别和测量水平的miRNA存在于外来外来。

<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>EDTA coated vacutainer (10 ml tubes)</td>
 <td>BD Diagnostics</td>
 <td>366643</td>
 <td>For exosome purification from human blood</td>
 </tr>
 <tr>
 <td>EDTA coated vacutainer (2 ml tubes)</td>
 <td>BD Diagnostics</td>
 <td>367841</td>
 <td>For exosome purification from mouse blood</td>
 </tr>
 <tr>
 <td>PAXgene Blood RNA Tube</td>
 <td>BD Diagnostics</td>
 <td>762165</td>
 <td>For miRNA isolation from total blood</td>
 </tr>
 <tr>
 <td>miRVana microRNA isolation kit</td>
 <td>Ambion</td>
 <td>AM1561</td>
 <td></td>
 </tr>
 <tr>
 <td>Acid-Phenol: CHCl3</td>
 <td>Ambion</td>
 <td>9721G</td>
 <td></td>
 </tr>
 <tr>
 <td>DNase 1</td>
 <td>Qiagen</td>
 <td>79254</td>
 <td></td>
 </tr>
 <tr>
 <td>TaqMan Universal PCR Master Mix, No AmpErase UNG</td>
 <td>Applied Biosystems</td>
 <td>4326614</td>
 <td>For TLDA cards</td>
 </tr>
 <tr>
 <td>Megaplex RT rodent Pool Set v3.0</td>
 <td>Applied Biosystems</td>
 <td>4444746</td>
 <td></td>
 </tr>
 <tr>
 <td>Megaplex preamp rodent Pool set v3.0</td>
 <td>Applied Biosystems</td>
 <td>4444747</td>
 <td></td>
 </tr>
 <tr>
 <td>Taqman Array Rodent MicroRNA A+B set V3.0</td>
 <td>Applied Biosystems</td>
 <td>4444909</td>
 <td></td>
 </tr>
 <tr>
 <td>Megaplex RT Human Pool set V3.0</td>
 <td>Applied Biosystems</td>
 <td>4444745</td>
 <td></td>
 </tr>
 <tr>
 <td>Megaplex Preamp human pool set v3.0</td>
 <td>Applied Biosystems</td>
 <td>4444748</td>
 <td></td>
 </tr>
 <tr>
 <td>Taqman Array Human MicroRNA A+B Cards Set v3.0</td>
 <td>Applied Biosystems</td>
 <td>4444913</td>
 <td></td>
 </tr>
 <tr>
 <td>Taqman MicroRNA RT kit</td>
 <td>Applied Biosystems</td>
 <td>4366596</td>
 <td></td>
 </tr>
 <tr>
 <td>Taqman fast universal PCR master mix </td>
 <td>Applied Biosystems</td>
 <td>4366072</td>
 <td>For mRNA qRT-PCR</td>
 </tr>
 <tr>
 <td>Tumor necrosis factor (primer probe)</td>
 <td>Applied Biosystems</td>
 <td>Hs01113624_g1 </td>
 <td></td>
 </tr>
 <tr>
 <td>Vascular endothelial growth factor A (primer probe)</td>
 <td>Applied Biosystems</td>
 <td>Hs00900055_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>Maxima First Strand cDNA Synthesis Kit for RT-qPCR</td>
 <td>Thermo Scientific</td>
 <td>K1642</td>
 <td>For mRNA</td>
 </tr>
 <tr>
 <td>HSP70 antibody</td>
 <td>Abcam</td>
 <td>ab94368 </td>
 <td>For western blot</td>
 </tr>
 <tr>
 <td>Anti-rabbit IgG-Gold</td>
 <td>Sigma</td>
 <td>G7402</td>
 <td>For electron microscopy</td>
 </tr>
 <tr>
 <td>Rabbit-anti CD81</td>
 <td>Sigma</td>
 <td>SAB3500454</td>
 <td></td>
 </tr>
 <tr>
 <td>Nickel 300 mesh carbon formvar grids</td>
 <td>Electron Microscopy Sciences</td>
 <td>FCF300-Ni</td>
 <td></td>
 </tr>
 <tr>
 <td>Copper 300 mesh carbon formvar grids</td>
 <td>Electron Microscopy Sciences</td>
 <td>FCF300-Cu</td>
 <td></td>
 </tr>
 <tr>
<td></td>
<td></td>
<td></td>
<td>Table 1. Table of specific reagents.</td>
</tr>
 </tbody>
 </table>

purification and microrna profiling of exosomes derived from blood and culture media

稳定的miRNA是目前在所有的体液和一些循环miRNA是封存在小囊，称为外来免受降解。外来体可以在RNA和蛋白质转移到目标小区与质膜融合。它们的生物学功能包括免疫反应，抗原呈递细胞内的通信。受体细胞通过血液，可以调节基因表达的miRNA交付开辟了新的途径为目标的干预。除了提供交付药物或RNA的治疗药物的战略，exosomal内容可以作为生物标志物，可以帮助诊断，确定治疗方案和预后。 在这里，我们将介绍的程序定量分析miRNA和外来的信使RNA（mRNA）的分泌在血液和细胞培养基。纯化的外来的特点将是使用免疫印迹分析EXOS的OMAL标记和PCR检测mRNA的兴趣。透射电子显微镜（TEM）和免疫金标记将被使用的，来验证exosomal的形态和完整性。从这些外来体将纯化总RNA，以确保我们可以从相同的样本研究的mRNA和miRNA。验证RNA的完整性，生物分析仪后，我们会进行一个中等通量定量实时PCR（qPCR）的使用Taqman探针低密度阵列卡（TLDA）和基因表达研究感兴趣的成绩单，识别exosomal miRNA的。 这些协议可以用于量化的患者中，啮齿动物模型和药物干预前，后的细胞培养基改变exosomal的miRNA。不同的Exosomal内容来源及细胞分泌外来体的生理条件。这些变化，可以了解单元和系统如何应对压力或生理扰动。我们的有代表性的数据展示Variations的miRNA纯化小鼠血液，人体血液和人体细胞的培养基中存在的外来。 在这里，我们将介绍的程序定量分析miRNA和外来的信使RNA（mRNA）的分泌在血液和细胞培养基。纯化的外来的特点将是感兴趣的mRNA的的exosomal标记和PCR使用免疫印迹分析。透射电子显微镜（TEM）和免疫金标记将被使用的，来验证exosomal的形态和完整性。从这些外来体将纯化总RNA，以确保我们可以从相同的样本研究的mRNA和miRNA。验证RNA的完整性，生物分析仪后，我们会进行一个中等通量定量实时PCR（qPCR）的使用Taqman探针低密度阵列卡（TLDA）和基因表达研究感兴趣的成绩单，识别exosomal miRNA的。 这些协议可以用来量化的改变，在exo​​somal miRNA的我Ñ​​患者，啮齿类动物模型，药物干预前后细胞培养基。不同的Exosomal内容来源及细胞分泌外来体的生理条件。这些变化，可以了解单元和系统如何应对压力或生理扰动。我们的有代表性的数据显示变化的miRNA存在于外来鼠标血，人体血液和人体细胞培养介质纯化

从血液或细胞培养介质隔离外来后，外来的纯度可以测试通过电子显微镜（EM）和免疫印迹（ 图1A和1B）。我们证实了我们的外切体的制剂， 图1A示出从各种来源与EM和使用多个抗体的免疫印迹。确认外来体具有直径约30 -100 nm处是完整的，并包含CD81的免疫金标记的EM图像。常用的exosomal标记是Hsp70和tetraspannin的家庭糖蛋白CD63，CD81和CD9 14。确认纯化的外?...

Watch this Scientific Journal Video about Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media at JoVE.com

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

存在稳定的microRNA（miRNA的）外来间通讯作为一种新型的模式产生了巨大的兴趣，为他们的潜在效用作为生物标志物和治疗干预的路由。在这里，我们展示了切体净化血液和文化传媒定量PCR确定的miRNA被运。

外来体的纯化和microRNA剖析来源于血液和文化传媒

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

purification-microrna-profiling-exosomes-derived-from-blood-culture

Research

JoVE Journal

Biology

31.7K 次观看.  Drexel University College of Medicine. 存在稳定的microRNA（miRNA的）外来间通讯作为一种新型的模式产生了巨大的兴趣，为他们的潜在效用作为生物标志物和治疗干预的路由。在这里，我们展示了切体净化血液和文化传媒定量PCR确定的miRNA被运。

视频: Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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 ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA含外来体的分离和鉴定

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

来自人类脐带血内皮细胞集落形成细胞的表型和功能特性

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

从人骨骼肌隔离血管源性多能前体

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

人类iPS细胞的微小RNA表达谱，视网膜色素上皮衍生自IPS和胎儿视网膜色素上皮

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

人外周血代文化血液的产物内皮细胞

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Purification of Platelets from Mouse Blood

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. We describe here purification of platelets from mouse blood using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 &#176;C. The recovery/yield of the purified platelets were 18.2-38.5%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets treated with thrombin showed up to 93% activation, indicating their viability. We confirmed that the purified platelets are sufficiently pure using flow cytometric and microscopic evaluation. These platelets can be used for gene expression, activation, granule release, aggregation, and adhesion assays. This method can be used for purification of platelets from the blood of other species as well.

Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.

小鼠血液中血小板的纯化

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), ...