这项研究是由瑞典研究理事会（K2008 - 57X - 20 676-01-3）的资助。

1。切体隔离<ol><li>增长切体无血清培养基的细胞。因此，任何血清细胞培养液中添加的外来应耗尽120万个晚上XG离心在4 ° C后才能使用。 </li><li>将细胞悬液锥形管。 </li><li>在4 ° C沉淀细胞，离心10分钟300 XG。 </li><li>转移上清超速离心机管，如果不彻底全面加入PBS。 </li><li>样品16 500 XG离心20分钟，4 ° C至进一步去除细胞和细胞碎片。 </li><li>通过0.2微米过滤除去大于200纳米的颗粒过滤上清液。 </li><li>过滤上清液转移到新的超速离心机管和120 000 XG 70分钟前，超速离心机密封管在4 ° C至颗粒的外来体。 </li><li>弃上清。 </li><li>最大的外切体检索，悬浮切体充实ED颗粒多次在一个适当的缓冲体积小（〜3 × 50μL）。这个缓冲区取决于以下的切体隔离计划的下游实验。例如，裂解液用于蛋白质和RNA的分离，PBS是用于电子显微镜和流式细胞仪和功能的研究介质可能是首选。 </li></ol>注;外来体也可以从不同的体液，如血浆，使用相同的过程，作为细胞培养基中分离。对于粘性液体，它可能是必要的，用PBS稀释的样品离心和过滤步骤之前的。在上述5点的离心速度，它可以增加至29 500 XG，第7点中的离心可以延长至90分钟18。 如果样品需要进一步纯化的外切体颗粒可漂浮在蔗糖梯度和外来将主要分数representing一个密度1.13-1.19克/毫升2。 2。切体识别电子显微镜<ol><li>为了进一步消除污染蛋白，重悬在120 000 XG 70分钟，在4 ° C至重新沉淀外来的外切体的丰富沉淀，PBS和超速离心机。 </li><li>的蛋白分离和总蛋白测定样品的小等分。确保只有一小部分样品裂解和蛋白质测量，并保存完整的外来体，解决的PBS，单独的冰或在-80℃为进一步的实验。 </li><li>将在PBS重悬的完整的外来exosomal蛋白质约10微克，一个下降，一个封口膜。然后，用血管钳，轻轻的位置上每一滴30-60分钟的顶部formvar碳包覆镍网格。保证网格定位涂层的一面面临下降含外来。 </li><li>将三滴，每30μL的PBS，在T他封口膜和洗网格，按顺序定位的PBS液滴上的电网和使用吸油纸之间。使用吸油纸轻轻只是抱着它不涂面积接触，密切合作，以电网侧。 </li><li>修复存款样品的封口膜下降了2％多聚甲醛，并放置在顶部的10分钟下降的网格。 </li><li>重复洗涤步骤中的第4点，前一个合适的抗体免疫。常用的抗体是抗CD63和抗MHCⅡ类。电子显微镜也可以不免疫作为外来体的大小和形态上，完全可以识别基于。但是，我们建议，评估的大小，形态和为一个更确凿的验证的膜蛋白的存在。 </li><li>网格传输选择的主要抗体30μL下降，孵育40分钟。洗净重复第4点，但在PB中使用0.1％的牛血清白蛋白S代替的PBS单独。 </li><li>重复第7点，但与10纳米金标记的第二抗体和单用PBS洗。 </li><li>修复后下降了2.5％戊二醛中加入的封口膜样品，孵育10分钟下降顶部的网格。重复第4点的洗，但使用去离子水液滴，而不是三个水滴的PBS。 </li><li>对比样品加入下降2％醋酸铀的封口膜，下降为15分钟的顶部，并培育网格。 </li><li>嵌入下降了0.13％甲基纤维素和0.4％的醋酸铀的封口膜样品，孵育10分钟下降顶部的网格。 </li><li>删除多余的液体，然后轻轻地使用吸油纸的定位与涂边格在一张纸上，让空气干燥5分钟。 </li><li>用电子显微镜检查的筹备工作，或存储在网格中的电网，为今后的工作。 </li></ol> 3。由流式细胞仪的外切体的表征作为外来太小，由目前的流式细胞仪设备检测，有必要先绑定外来抗体包被的磁珠（ 图1） 。这些珠子可以购买一个现成的产品，或选择的抗体在实验室。根据exosomal细胞来源，不同的抗体可以配上珠可以磁或乳胶字符。我们目前使用的抗MHC II类或抗CD63的包被的磁珠，这种分析。 <ol><li>对于使用"国产"的抗体包被的磁珠，我们使用4微米乳胶具有抗CD63抗体包被的磁珠。 25μL4微米乳胶珠（30 × 10 6珠）100μL的MES缓冲的两倍，3 000 XG 15-20分钟，洗净，在100μL的MES缓冲溶解沉淀。准备抗体的混合物，其中包含一个体积等于12.5微克抗体与相同体积的MES缓冲。添加珠抗体的混合物，在室温下孵育过夜搅拌。抗体包被的磁珠洗净，用PBS（3 000 20分钟XG）三次沉淀溶于100μL存储缓冲区（与终浓度为300 000珠/μL）。 </li><li>对于每个样本（每个抗体），继续与卷等于30微克exosomal蛋白质的最低％〜100 000抗体包被珠（PBS中解决了完整的外来）。 </li><li>孵育外来珠，在一个总体积300μLPBS过夜，在4 ° C下轻柔的动作。 </li><li>加入300μL200毫米甘氨酸和孵化30分钟座。 </li><li>两次洗涤缓冲液的切体珠复合物（1-3％血清的PBS），10分钟600 XG洗净。 </li><li>孵育50μLIgG抗体的外切体珠复合物4 ° C。在洗涤缓冲液洗净切体珠复合物，在第5步中所述的两倍。 </li><li>添加90μL洗涤缓冲液和10μL抗体的选择（理想的抗CD9，抗CD63或CD81的反）的切体珠复合物和40分钟，轻柔的动作下孵育。在洗涤缓冲液的切体珠复合物在第5步中所述的两次洗净。 </li><li>加入300μl洗涤缓冲液，并用流式细胞仪采集数据。 </li></ol>如上所述，不同的珠子可以使用，如协议或磁珠所述的乳胶珠。如果用来代替磁珠，请注意，该协议是略有不同。阻塞步骤（4点）是不是必需的，是由放置在一个磁性的立场，而不是一个离心管进行洗。 "自制"的抗体包被的磁珠使用的存储缓冲区，包含0.1％甘氨酸和azid在PBS，pH值7.2，0.1％钠。 更重要的是，确保使用流式细胞仪洗涤缓冲液（1-3％血清的PBS）外切体耗尽血清，以避免任何血清外来污染分析。 请注意，使用抗体加上珠的切体的表征时，重要的是要承认，这仅仅是一个特定的亚群，特定于所使用的抗体，即分离并鉴定。 4。切体检测用Western blot Western印迹是一个行之有效的方法，我们不会细讲就方法本身，但重点放在一个合适的蛋白分离前的印迹和使用不同的抗体的重要性。 <ol><li>溶解蛋白裂解液的选择外切体颗粒，并彻底吹打，旋涡混合。要进一步裂解的外来体，超声水浴中3 × 5分钟之间的旋涡混合样品。最后，在室温下5分钟离心样品13 000 XG，将上清转移到一个新的Eppendorf管。 </li><li> Measu重新选择的方法总蛋白和负载20-50微克的蛋白质，每口井。 </li><li>凝胶电泳和电泳分离和转移的蛋白质。 </li><li>座和洗膜，然后再执行对在外来丰富的蛋白质免疫。 </li><li>检测化学发光数字成像系统和分析软件的特定蛋白质。 </li></ol>至于有没有切体的特异性标志物，在外来丰富的蛋白质，从各个不同的细胞起源，是常用的外切体检测。这些tetraspanins（如CD9，CD63和CD81），细胞骨架相关蛋白（如ezrin）和多泡的生物合成（如TSG101和ALIX）涉及的蛋白质，如蛋白质。其他蛋白质也常用于外来检测Flotillin，HSC70和不同的Rab蛋白等，但也有特定的蛋白质，如A33（肠上皮细胞）的外来细胞中CD3（T细胞）和MHCⅡ类（抗原呈递细胞）。因此，根据外来体是由什么类型的细胞进行检测标记发布可能会有所不同。 由于其他车厢的细胞也能产生水泡，这是进一步建议，以确定这些车厢，如内质网（如calnexin和GRP78）和高尔基体（如GM130）的蛋白质的存在。因此，这些蛋白质的缺乏表示没有或很少污染样品中的其他车厢的囊泡研究。 5。 exosomal RNA和RNA分析的分离<ol><li>切体颗粒溶解在RNA裂解液的选择和执行RNA的提取。根据下游实验不同的RNA分离试剂盒可用于，但基于列的方法提供了更广泛的RNA样品。目前，我们正在使用由Exiqon miRCURY RNA分离试剂盒，但可能适合在SC上其他套件ientific问题就在眼前。 </li><li>在进行RNA的分离是很重要的工作在无RNase的环境。因此，使用核酸和核酸免费枪头和擦拭工作台和设备，无污染物和核糖核酸酶。 </li><li> RNA的提取，使用miRCURY RNA提取试剂盒，溶于350μL裂解液的切体颗粒，并执行旋涡混合15秒。 </li><li>加入200μl95％乙醇和10秒的旋涡混合的。 </li><li>放置在一个收集管一列，并转移裂解外来列和离心1分钟14 000 X G. </li><li>包含exosomal RNA和离心1分钟14 000 X G.列加入400μL洗涤液洗三次</li><li>离心2分钟14 000 XG，以确保该列是干的，并放置在一个无RNase Eppendorf管干栏列。 </li><li>加入50μL洗脱液列和2型离心机<html> 200 XG分钟，其次是1分钟14 000 X G. </li><li>继续分离出的RNA，或存放在-80 ° C。 </li></ol>有关检测和分析，提取总RNA的exosomal的，使用的RNA 6000 Nano或RNA 6000碧套件，安捷伦2100生物分析仪。分析表明exosomal RNA的产量和粒度分布。 6。代表性的成果外来体是太小，可用流式细胞仪方法检测，并因此连接，然后分析抗体包被的磁珠（ 图1） 。切体珠复合物可聚集流式细胞仪散点图可以包含不同人群的单人，双人和三珠（ 图2A） 。箭头指示，进一步分析单珠。一个CD63阳性单珠切体的人口相比，它的同型对照如图 2b所示。 在图3中 ，电子显微镜e_content"&gt;由于外来的小规模，他们只能直接用电子显微镜观察，而不是通过光镜，典型的外来体的形态特征是圆形和30-100纳米大小的膜泡。照片显示，外来体免疫染色与金标记抗体CD63（箭头指示）和非免疫染色外来（二）（ 一 ） 。 要确定确实是孤立的囊泡外来进一步刻画exosomal蛋白质，免疫印迹常用图4显示了calnexin的情况下，内质网标记。这表明从内质网囊泡小污染。此外， 图4显示的CD81的外来体的存在，这是通常在外来丰富tetraspanin 。 RNA的主要含有小号核糖体RNA（rRNA的），在生物分析仪分析（ 图5A）为18S和28S rRNA的亚基的两个突出的峰看到。然而，exosomal RNA的不同在他们的个人资料，作为外来缺乏rRNA的两个峰（18S和28S）和丰富的mRNA和miRNA（ 图 5B），如短RNA。 <img alt="图1" src="/files/ftp_upload/3037/3037fig1.jpg" /> 图1。由于外来（30-100纳米）的体积小，他们不能区分噪声和背景时，流式细胞仪分析。因此，有必要将其连接到抗体包被的磁珠的外来之前进行了分析，然后可以由激光显示。 <img alt="图2" src="/files/ftp_upload/3037/3037fig2.jpg" /> 图2。珠的切体复合物可与对方和形式的双重和三重珠聚合（一）。箭头证明单珠，出<html>这是gated和用于进一步分析的一些人口。这个人口将直接相比，同型对照（充满灰色峰）来判断膜的外来分子的存在。 CD63的积极外来体（黑色开峰） 如图 B所示。 <img alt="图3" src="/files/ftp_upload/3037/3037fig3.jpg" /> 图3。外来的电子照片，与典型的形态和大小（40-80纳米）（A和B ）。图中的箭头 A表示二级抗体的黄金粒子，验证，这种切体有其膜表面CD63的。 <img alt="图4" src="/files/ftp_upload/3037/3037fig4.jpg" /> 图4。 Western blot结果显示内质网蛋白calnexin但常用的外来体，tetraspanin CD81的丰富的蛋白质存在的情况下。 ve_content"&gt; <img alt="图5" src="/files/ftp_upload/3037/3037fig5.jpg" /> 图5。生物分析结果显示存在的RNA在细胞（A）和外来（二）。箭头表示18S和28S rRNA的亚基在细胞中存在。由于exosomal RNA的男子气概包含小分子RNA，没有或很少rRNA的发现外来体。

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Cell Biol. 9, 654-659 (2007).</li><li>Eldh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+communicate+protective+messages+during+oxidative+stress;+possible+role+of+exosomal+shuttle+RNA.">Exosomes communicate protective messages during oxidative stress; possible role of exosomal shuttle RNA.</a> PLoS One. 5, e15353-e15353 (2010).</li><li>L&#228;sser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+saliva,+plasma+and+breast+milk+exosomes+contain+RNA:+uptake+by+macrophages.">Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages.</a> J. Transl. Med. 9, 9-9 (2011).</li></ol>

巨浪是利用为载体的RNA转移到细胞的外来体相关专利的共同拥有。

在研究分子的内容和/或外来体的功能，它使用高品质的隔离方法，提供了一个适当的产量和尽可能低的程度的污染，是至关重要的。本文中所描述的方法是行之有效的方法为隔离外来和研究他们的RNA含量和生物功能。此外，隔离外来的特性的重要性，由不同的方法，包括电子显微镜，流式细胞仪和Western blot，相结合，突出。 隔离外来体时，首先离心的步骤（300 XG）用于细胞悬液或研究的生物流体中的颗粒细胞可能。第二离心16 500 XG需要强大到足以沉淀的死细胞，较大的碎片，凋亡小体和其他细胞器。在此之后离心，一些协议，包括纳米过滤步骤，但一些INVestigators已经排除了这一步。然而，我们强调消除碎片和囊泡大于200纳米，因此强烈建议包括一个过滤步骤的重要性。如果需要更严格的隔离，可用于100纳米过滤器，但要注意，如果使用粘性流体，这些过滤器可以很容易成为阻塞和exosomal材料丢失。另外，100纳米过滤可能影响的外来的形态，此外，如果外来规模较大的是，他们可能会丢失。因此，200纳米过滤器似乎适合切体隔离。 120000 XG强制离心过滤后，用于颗粒的外来。外来可以进一步用PBS冲洗，势必抗体包被的磁珠洗，或放置在蔗糖梯度消除污染蛋白质。然而，这可以是一个非常消耗资源的过程中，我们认为没有必要，特别是如果起始样品体积小和/或RNA的内容f的外来体是低的，额外的步骤，将大幅减少进一步的材料。然而，孤立的囊泡的性质应证明的存在和缺乏某些蛋白质。 迄今为止，没有外来的绝对独特的蛋白标记已经确定，以确认样品中含有外来体，并没有别的。因此，需要相结合的方法来刻画外来体，包括确定的规模和形态的电子显微镜的外来。此外，样品的纯度可通过电子显微镜确定，这种方法可以提供较大的囊泡，如微粒，凋亡小体或细胞碎片，概述样品的污染程度，例如，。此外，在外来的蛋白质含量可确定通过流式细胞仪和Western blot，这两种方法的结果在分析双方的膜结合的约束（如CD63的CD81）和内在蛋白（如Tsg101基因和阿利克斯）的外来体。检测外来体，如CD63，TSG101和阿利克斯，缺乏蛋白质，如内质网蛋白calnexin丰富的蛋白质，是一个迹象表明，丰富的外切体颗粒的确是外来的和不污染其他车厢的细胞囊泡。 外来检测RNA的配置文件，在完整的细胞中发现的RNA是根本不同的。因此，生物分析仪或凝胶为基础的RNA分析方法分析exosomal RNA，缺乏为18S和28S rRNA的亚基，这是在分析RNA的突出两峰。因此，我们认为，rRNA的任何样品中的大量的检测表明，提取总RNA exosomal起源并不孤单，从而隔离的方法，需要加以改进和质量保证。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> 切体隔离 </td></tr><tr><td>的Optima L - 90K超速离心机</td><td> Beckman Coulter公司</td><td> 65670 </td><td></td></tr><tr><td>快速密封超速离心机管</td><td> Beckman Coulter公司</td><td></td><td>根据转子采用了不同的管是必要的。 Ti70（342414），Ti45（345776） </td></tr><tr><td>快速密封无绳管礼帽套件</td><td> Beckman Coulter公司</td><td> 358312 </td><td></td></tr><tr><td> Filtropur注射器过滤，0.20μm </td><td> Sarstedt </td><td> 83.1826.001 </td><td>对于粘胶液体和/或大批量，真空过滤系统，0.2微米VWR International的，可以用来代替（514-0600）。 </td></tr><tr><td> 电子显微镜 </td></tr><tr><td> Formvar /碳包覆镍电网</td><td>泰德佩拉公司</td><td> 1GN200 </td><td></td></tr><tr><td>鼠抗人CD63的</td><td> BD生物科学</td><td> 556019 </td><td>终浓度:0.05μg/μl </td></tr><tr><td>同型对照;IgG1κ（MOPC 21） </td><td> Sigma - Aldrich公司</td><td> M9269 </td><td>终浓度:0.05μg/μl </td></tr><tr><td>抗鼠IgG，10纳米黄金共轭</td><td> Sigma - Aldrich公司</td><td> G7777 </td><td>终浓度:1％ </td></tr><tr><td> LEO 912AB欧米茄电子显微镜</td><td>卡尔蔡司新界南总区</td><td></td><td>或同等设备。 </td></tr><tr><td> 流式细胞仪 </td></tr><tr><td> 4μm的aldehyd /硫酸乳胶珠</td><td>界面动态</td><td> 12-4000 </td><td></td></tr><tr><td>涂料的乳胶珠的抗体</td><td></td><td></td><td>例如抗- CD63的自BD生物科学（556019） </td></tr><tr><td>智能混音器RM - 2L </td><td> ELMI </td><td></td><td>或同等设备。 </td></tr><tr><td>从人类血清抗体</td><td> Sigma - Aldrich公司</td><td> I4506 </td><td></td></tr><tr><td>抗体检测</td><td> BD生物科学</td><td></td><td>例如:PE抗CD63（556020）PE抗CD9（555372）PE抗CD81的（555676），同型对照（555749） </td></tr><tr><td>屋宇署FACSAria </td><td> BD生物科学</td><td></td><td></td></tr><tr><td> 免疫印迹 </td></tr><tr><td> Transsonic T 490卫生署</td><td> ELMA </td><td></td><td>或同等设备。 </td></tr><tr><td> exosomal RNA和RNA分析的分离</td></tr><tr><td> miRCURY的RNA分离试剂盒</td><td> Exiqon A / S公司</td><td> 300110 </td><td></td></tr><tr><td>安捷伦2100生物分析仪</td><td>安捷伦科技公司</td><td> G2939A </td><td></td></tr></tbody></table>

isolation and characterization of rna-containing exosomes

切体研究领域正在迅速扩大，在出版物近年来急剧增加。这些小囊泡内吞的起源（30-100纳米）首先提出功能的一种方式为网织红细胞，以消除转铁蛋白受体，而成熟红细胞 1，后来被命名为外来。外来体是由外来萌芽后期内涵体产生多泡机构（MVBs），形成，并释放到环境中的融合与质膜 2 MVBs 。首次发现的外来以来，一个广泛的细胞已被证明释放这些囊泡。外来体也已在几种生物体液，包括血浆，鼻腔灌洗液，唾液和乳汁3-6中检测到。此外，它已被证明外来的内容和功能取决于原始细胞和在何种情况下他们的生产条件。多种功能已被恶魔trated为外来体，如诱导对过敏原7,8，建立肿瘤根除小鼠9，激活自然杀伤细胞的抑制和10-12，促进分化为调节性T细胞，刺激T细胞增殖 14的耐受性和诱导T细胞凋亡15。 2007年，我们证明，从肥大细胞释放的外来包含信使RNA（mRNA）和microRNA（miRNA）的，和RNA可以从一个细胞到另一个通过外来穿梭。在受体细胞中，外来穿梭的mRNA被翻译成蛋白质，表明监管职能的转移核糖核酸 16 。此外，我们还表明，氧化胁迫下生长的细胞派生的外来可诱导对进一步强调在受体细胞耐受性， 因此建议的17 exosomal穿梭RNA的生物学功能。细胞培养和生物体液中的合作ntain囊泡的混合物，并流下片段。一个高品质的外来隔离方法，由外来，其内容的表征和鉴定，因此，以区别于其他的囊泡和颗粒的外来至关重要。在这里，我们目前的隔离从细胞培养液和体液的外来的方法。这种隔离方法的基础上反复离心，过滤步骤，最后的离心步骤，在其中的外来颗粒。突出重要的方法来识别外来和exosomal形态和蛋白质含量的特征，包括电子显微镜，流式细胞仪和免疫印迹。总exosomal RNA纯化是基于自旋柱层析和exosomal RNA的产量和粒度分布分析一个生物分析仪。

Watch this Scientific Journal Video about Isolation and Characterization of RNA-Containing Exosomes at JoVE.com

Isolation and Characterization of RNA-Containing Exosomes

本文演示的外来隔离，净化和检测，以及其分子的内容分析技术的方法。这些方法适应切体细胞培养基和生物体液隔离，并可以分析分子的内容以外，还可以在功能的研究非常有用。

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-characterization-of-rna-containing-exosomes

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

99.3K 次观看.  University of Gothenburg. 本文演示的外来隔离，净化和检测，以及其分子的内容分析技术的方法。这些方法适应切体细胞培养基和生物体液隔离，并可以分析分子的内容以外，还可以在功能的研究非常有用。

视频: Isolation and Characterization of RNA-Containing Exosomes

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

改造使用含RNA寡核苷酸基因组DNA的RNA / DNA杂交一代

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

科研

生物学

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

对赫斯特的分离与表征低 CD45负小鼠肺间质干细胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

分离，培养和成年小鼠Cardiomyoctyes的功能分析

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Characterization of Microvesicles from Peripheral Blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

从外周血中分离和鉴定的微泡

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心脏纤维细胞和肌纤维细胞的分离与表征

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

骨骼肌成纤维细胞外泌体的分离和表征

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

小鼠原发性肝正弦内皮细胞的分离与表征

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Isolation and Characterization of Intact Phycobilisome in Cyanobacteria

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. Studying the structure and composition of phycobilisome is of great interest to scientists because it reveals the evolution and divergence of photosynthesis in cyanobacteria. This protocol provides a detailed and optimized method to break cyanobacterial cells at low cost by a bead-beater efficiently. The intact phycobilisome can then be isolated from the cell extract by sucrose gradient ultracentrifugation. This method has demonstrated being suitable for both model and non-model cyanobacteria with different cell types. A step-by-step procedure is also provided to confirm the integrity and property of phycobiliproteins by 77K fluorescence spectroscopy and SDS-PAGE stained by zinc sulfate and Coomassie Blue. The isolated phycobilisome can also be subjected to further structural and compositional analyses. Overall, this protocol provides a helpful starting guide that allows researchers unfamiliar with cyanobacteria to quickly isolate and characterize intact phycobilisome.

The current protocol details the isolation of phycobilisomes from cyanobacteria by centrifugation through a discontinuous sucrose density gradient. The fractions of intact phycobilisomes are confirmed by 77K fluorescent emission spectrum and SDS-PAGE analysis. The resulting phycobilisome fractions are suitable for negative staining of TEM and mass spectrometry analysis.

蓝藻中完整藻胆体的分离与表征

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. ...

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models. Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles -&#160;small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis. Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.

The present protocol providesdetailed descriptions for isolation, concentration,and characterization of extracellular vesicles from cyanobacterial cultures. Approaches for purifying vesicles from cultures at different scales, trade-offs among methodologies, and considerations for working with field samples are also discussed.

RNA含外来体的分离和鉴定

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