The authors acknowledge Meike Schaffrinski for excellent technical assistance. We would like to thank the following people for their help in the collection of peripheral blood samples (all from University Medical Center G&#246;ttingen): Henrietta Vida (Dept. of Transfusion Medicine), Kia Homayounfar, Lena-Christin Conradi (Dept. of General, Visceral and Pediatric Surgery), Leila Siam, Bawarjan Schatlo (Dept. of Neurosurgery), Hendrik A. Wolff, Martin Canis (Dept. of Otorhinolaryngology, Head and Neck Surgery) as well as all employees of the interdisciplinary short-term oncology. We acknowledge Dirk Wenzel (Max Planck Institute for Biophysical Chemistry, G&#246;ttingen) for his help with the electron microscopy of MVs. 
The study was funded by the German Ministry of Education and Research (BMBF) project MetastaSys (grant no. 0316173) as well as the German Cancer Aid (grant no. 109615).

所有的实验，包括人类受试者已批准由当地伦理委员会（批准文号14年3月2日）。对患者的选择应当注意的是，几个因素，如年龄，性别，当前治疗方案和许多可能影响血液的MV组合物，因此，应当考虑到事先考虑到样品采集22,23。 1.血浆样品的制备<ol><li>通过21号蝴蝶针到含有EDTA（1.6毫克/毫升血）一个真空采血管的血液收集管的每个供体血2管 - 绘制1。确保反转管（S）数次，以保证有效的抗凝血。 注意:血后续流式细胞仪和Western blot分析推荐量是5 - 15毫升。为了防止压退化和丧失，血样应采血后30分钟进行处理&lt;。 </li><li>离心15分钟将样品在1200 xg离心制备血浆样品室温（RT）。 </li><li>为了从剩余的血细胞（=下层）帮助等离子体（=上层）的分离应用阀的过滤器。 </li><li>等离子体转移到15毫升管中。 </li><li>在1500 xg离心离心15分钟，室温以沉淀较大细胞碎片和除去残留的血小板。 </li><li>将上清转移到15毫升管中并用中压分离或商店的样品直接进行在-20℃下长达6个月。 注意:所提出的协议，也可以用于分离从细胞培养物上清液的MV（和的Exos）。为了做到这一点，培养细胞在60％ - 80％汇合24 - 在培养基中48小时补充有囊泡贫FCS，然后回收上清。离心机在750 xg离心5分钟，4℃至耗尽残余漂浮细胞，再次填充上清液到新的15毫升管中并离心5分钟，在1500 xg离心，4℃以沉淀较大细胞碎片。然后该上清液可用于MV的隔离2.16 - 在2.1步骤描述。 </li></ol> 2. MV的隔离<ol><li>在一个合适的离心管转移血浆样品。如果需要的话，为了在离心分离过程中稀释样品，并防止薄壁管的塌陷填充管，用PBS。 </li><li>离心机在14000 xg离心，4℃35分钟。 </li><li>倒出上清液，保持管颠倒了，把在纸巾上。等待3 - 5分钟，直到所有剩余的上清液已经浸透到毛巾，从而从样品中除去。 </li><li>重悬在1000微升PBS中的MV沉淀，转移到1.5mL管中，并离心35分钟在14000 xg离心，4℃下在台式离心机。 </li><li>吸上清。 </li><li>重悬在50的MV粒料 - 500微升PBS中，取决于粒料的大小。备选地，裂解的MV直接， 例如 ，在RIPA缓冲液（150毫摩尔NaCl / 0.1％SDS / 0.5％的Na-脱氧胆酸盐/ 1％的Triton X-100 /的50mM Tris，pH7.2）中随后的免疫印迹分析。商店的MV在-20℃。他们将保持稳定数月，但要避免反复冻融周期。 </li><li>可选的:为了评估MV产量或剂量对于随后的实验的MV确定与蛋白测定的MV蛋白质浓度（ 例如 ，布拉德福德或Lowry法）。 </li><li>如果额外的Exos要从血浆样品中分离，滗析从步骤2.3上清液到超速离心管中，离心分离2小时在11万xg离心，4℃。如步骤2.3中所述倒出上清液，重悬外切粒料在1000微升PBS中，并转移到小（1.5mL）中超速离心管中。 <ol><li>超速离心2小时在11万xg离心，4℃，吸上清，重悬外切粒料在50 - 75微升PBS或RIPA缓冲液。 </li></ol></li></ol> 3.流式细胞仪MV的表征<ol><li>转移15微升PBS + 1％囊泡 - 贫胎牛血清（FCS）在流式细胞术管。 注:囊泡贫FCS由如先前24描述的（在56℃，30分钟）FCS的110000 XG通过0.2微米的过滤离心热灭活18小时，过滤上清液制备。 </li><li>添加5微克（在低收率3微克也适用的情况下）在PBS中的MV。 </li><li>孵育以阻断在压表面的非特异性的结合部位，从而减少背景染色在室温30分钟的样品。 </li><li>添加荧光标记的抗体对蛋白质的感兴趣。滴定抗体的用于为了确定最佳的浓度，并保证一个低信号对噪声比使用前染色的量。确保也包括一个管与未染色的MV作为阴性对照，并在同一浓度与匹配的同种型对照抗体染色的（ 例如 ，如果使用抗体的1微克，也使用同种型对照的1微克MV的一个管tibody）量化背景染色。 注意:也可以通过增加耦合到不同的荧光染料多种抗体流式细胞执行多色流。 </li><li>在室温下孵育在黑暗中20分钟。 </li><li>添加250微升PBS中，并使用流式细胞仪的试样的测定进行。 <ol><li>万一该样品不能被立即测量，添加150μL的PBS中，并加入50μL4％低聚甲醛（PFA）在4℃下以固定的样品和存储。注意:PFA是有毒的。使用手套和适当的个人防护装备。 </li></ol></li><li>减少流动的阈血细胞计数到可能的最低值，并搜索使用前向散射（FSC）对对数尺度侧散射（SSC）情节的MV人口。门上的MV人口并评估在相应直方图中的荧光信号。 </li></ol> 4.表征的MV由Western印迹<ol><li>直接悬浮颗粒MV中合适的裂解缓冲液（ 例如 ，RIPA缓冲液）。 <ol><li>在的情况下的MV粒料已经被重新悬浮在PBS中，稀释它至少1:1的在一个合适的裂解缓冲液（ 例如 ，RIPA缓冲液）。 </li></ol></li><li>确定的MV样品的蛋白质浓度， 例如，通过Lowry法。 </li><li>准备10 - MV的20微克22.5微升RIPA缓冲。然后在95℃下添加7.5微升4倍的Laemmli负载缓冲液和热5分钟。 </li><li>上的聚丙烯酰胺凝胶负载样品和根据标准方法进行电泳和免疫印迹。 </li><li>蛋白质转移到膜后，根据标准方法进行丽春红染色作为上样对照。 </li><li>在TBST脱色膜在RT 5分钟。 </li><li>块膜用于在TBST的5％BSA的30分钟到1小时在室温。 </li><li>在4℃下与第一抗体孵育膜过夜或在室温下2小时。 </li><li>用TBST 3×5分钟洗膜。</li><li>以1:1的稀释孵育与HRP偶联的二抗膜:在5 10,000％BSA中。注意:在高背景信号的情况下，使用奶粉代替BSA中。 </li><li>洗用TBST 3×5分钟的膜。 </li><li>与ECL检测试剂开发膜与化学发光膜或化学发光成像系统检测信号。 注意:为了区分从的Exos的MV，如微管蛋白的蛋白质，辅肌动蛋白-4或mitofilin可以使用哪些应主要存在于的MV 16,25。注意该用作的Exos标记最四旋抗体（ 例如，CD9，CD81），不减少的条件下工作，因此应在非还原加样缓冲液，然后加热10分钟在70℃来制备。 </li></ol>

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The authors have nothing to disclose.

<html>在血液电动车最近的研究已经表明，EV组合物和计数几种疾病的病因时改变。因此，这些电动车的分析和进一步特性是高息，以进一步评估疾病生物标志物诊断和预后的潜在用途或评估治疗的反应。我们在这里提出的协议允许囊泡的直径不包含任何细胞器达600纳米的隔离。这些观测结果与MV的当前定义行和排除凋亡小体2的存在。使用Western印迹，我们能够证明所述分离的MV显示微管蛋白的高表达，而通常用作外切标志物的tetraspanins CD9和CD81只略微表达。这证实了从的MV不同的Exos和蛋白质组学25装配到最近深入表征和两个EV人口比较。 内容"&gt;采集血液样本的过程中，关键是要以防止压降解尽可能短保持静脉穿刺和血浆制剂之间的时间。此外，血液样本的长期储存会导致血细胞造成增强MV的激活脱落和最终的细胞凋亡导致凋亡小体的释放。中压隔离的另一个重要的考虑是防止压制剂的污染与血浆蛋白或更小的Exos。因此，重要的是纺丝关闭之后，除去尽可能多的上清液尽可能在14000×g下的运动矢量。由于粒料通常是可见的并紧密附着到管的壁，将上清液可以容易地用移液管尖端除去。与此相反的Exos倾向于由高速超速离心并在制备过程中形成聚集体往往很难悬浮，不会的MV出现此问题。 我们的研究表明，它是possiBLE表征通过流式细胞仪存在于血液MV亚群。虽然大多数流式细胞仪的检测限为大约200 - 300 nm的，MVS分别重复地在供体以及与分析的相同的参数和门，清楚地允许其区别从背景信号细胞培养物的样品进行测定。它之前的PBS用于分析不含任何污染颗粒可能导致流式细胞术（ 图3）中一高背景测量，以验证是重要的。虽然一些较小的MV可能无法在流式细胞方法被捕获，检测来自所有主要血细胞种群（ 例如 ，血小板，红细胞，白细胞，内皮细胞）的MV。在我们的分析中，我们使用标准的标志物已在MVS 26先前发现不同的血细胞- 29。应当指出的是，为了获得流动的最佳可能结果术，叔他构成与所有抗体的浓度应在表达感兴趣抗原的MV样品进行滴定。如果血液中特定的MV亚群应具有更高的特异性来识别，也可以在各MV的针对两种不同的抗原本进行双染色，仅考虑所有双阳性的MV用于随后分析23。目前，有努力，在健康人23,30的血液来定义一个标准范围MV亚群。这些研究已经表明，血小板衍生的MV构成血液，这与我们的观察对应的MV的人口最多。 流式细胞术表征的MV样品的一个优点是，这种方法已经很好，在大多数临床中心诊断目的这将允许可能使用MV的如在日常临床诊断的生物标记物确定。在血电动车以往的研究具有多集中编在较小的Exos，依靠任何特定排序程序以选择性分析所需的外切目标人口31,32或需要耗时的（2天）的隔离带的Exos的耦合过程中胶乳分析前18小珠。我们自己未发表意见认为，从全血制品的MV的流式细胞仪分析甚至允许的MV的检测，如肿瘤衍生的MV没有任何这样的选择过程。 两者合计，这里介绍的协议允许MV的快速分离自外周血的样品与标准的实验室设备，并使用他们的后续表征流式细胞术和Western印迹。整个过程只需大约2小时，这将有利于今后对患者血液MV型材所需评估的MV的潜力疾病生物标志的研究进行。

在过去几年中的许多体外研究已经表明，细胞外囊泡（电动汽车）发挥细胞间通讯起重要作用。活细胞不断脱落它在规模，内容和生源差异囊泡。研究得最好的电动汽车是从该内体系统起源在那里它们被存储在多泡体腔内囊泡外来体。一旦与质膜后者保险丝，所包含的囊泡释放外来体（的Exos，直径30 - 100纳米1）。它已经获得了越来越多的关注在过去几年EV的第二人口大微泡（MVS，直径为100 - 1000纳米）从质膜2出芽直接。 这两种类型的小泡是由脂质双层包围和含有核酸， 例如DNA，mRNA或miRNA的3 - 5，以及它们可以转移到邻近的ç蛋白质的过多厄尔。而在一般的小泡的蛋白质组成反映原籍小区的状态，一些蛋白质似乎被选择性靶向和富集于电动汽车1。一个主要的研究兴趣是表征为了确定特定的EV签名可能允许使用电动汽车的作为新型生物标志物从异常和疾病细胞电动汽车。特别是在癌症，其中经常肿瘤本身不易获得，液体活检血液靶向肿瘤特异性电动车可能允许的治疗反应监测或帮助，而不需要侵入性程序6表征原发肿瘤。 事实上，电动汽车已成功地从各种体液，包括尿7，脑脊液8，母乳9或血液10隔离。几项研究确定了不同的人类疾病的EV数量的变化和组成。例如，在败血症患者的促凝的MV的数目是与健康个体相比11显著增加。此外，在重症患者脑型疟疾增加血液中总的MV可以观察和血小板衍生的MV计数昏迷深度和血小板减少12无关。其他研究患者系统性红斑狼疮或心脏衰竭而在后者的情况下，报告内皮衍生囊泡升高数字，这与心血管事件13,14的较高概率相关。 特别是在肿瘤，血液中的电动汽车正在与诊断和预后价值新型生物标志物进行讨论。表达肿瘤相关蛋白如MUC1，EGFR或FAK MV的水平似乎在乳腺癌患者15,16的血液中升高。还对的Exos，最近的研究已经表明，血液衍生的Exos携带肿瘤特异性抗原如磷脂酰肌醇聚糖-1为胰腺癌或德尔-1乳腺癌允许早期疾病德tection具有较高的特异性和敏感性17,18。此外，血清衍生肿瘤的Exos可以含有DNA，其可用于检测突变如KRAS和p53这表明其用于治疗的预测19使用。最近的进展已经表明使用特定的微流控芯片中胶质母细胞瘤患者的血液的Exos的该分析允许治疗20的监控。总之，这些发现意味着囊泡的疾病特异性亚群的分析提供了关于诊断，预后以及治疗选择和成功的有价值的信息。 然而，从血液的Exos的分离和分析是费时，需要特殊的实验室设备，因此，还没有适合于常规临床诊断。与此相反，MVS可更快分离，由于其较大的尺寸，可以通过流动容易地分析仪，而无需将它们耦合到乳胶珠，因为它是必要的Exos 1821。因此，这里我们提出了可用于MV的从血液样品标准化隔离并通过流式细胞术的MV亚种群的后续表征的协议。该协议将允许进一​​步的研究和对大型患者群体MV配置文件，将依次用于日常临床诊断的MV需要深入刻画。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>butterfly needle (21 gauge)</td>
			<td>Hospira Deutschland GmbH</td>
			<td>490P29201</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin Fraction V</td>
			<td>Roth</td>
			<td>8076.3</td>
			<td>For 10x TBS weigh 24.2 g Tris base and 80 g NaCl, ad 1 L H2O and adjust pH to 7.6</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>For 1x TBS-T mix 100 mL 10x TBS with 900 mL H2O and add 1 mL Tween-20</td>
		</tr>
		<tr>
			<td>CD235a-PE</td>
			<td>Beckman Coulter</td>
			<td>A07792</td>
			<td>use 5&#181;l for staining</td>
		</tr>
		<tr>
			<td>CD45-FITC</td>
			<td>Beckman Coulter</td>
			<td>7782</td>
			<td>use 5&#181;l for staining</td>
		</tr>
		<tr>
			<td>CD62E-PE</td>
			<td>Biolegend</td>
			<td>336008</td>
			<td>use 0.8&#181;g for staining</td>
		</tr>
		<tr>
			<td>CD62P-PE</td>
			<td>Biolegend</td>
			<td>304905</td>
			<td>use 0.1&#181;g for staining</td>
		</tr>
		<tr>
			<td>CD81 antibody</td>
			<td>Biolegend</td>
			<td>349501</td>
			<td>1:2000 in 5%BSA in TBS-T; use non-reducing conditions for Western Blotting</td>
		</tr>
		<tr>
			<td>CD9 antibody</td>
			<td>Immunotools</td>
			<td>21270091</td>
			<td>1:1000 in 5%BSA in TBS-T; use non-reducing conditions for Western Blotting</td>
		</tr>
		<tr>
			<td>Chemiluminescence imager ImageQuant LAS-4000</td>
			<td>Fujitsu Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DC protein assay kit II</td>
			<td>Bio-Rad</td>
			<td>5000112</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL detection reagent</td>
			<td>GE Healthcare</td>
			<td>RPN2232</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA vacutainers for blood collection</td>
			<td>Sarstedt</td>
			<td>01.1605.001</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSCanto II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fetal calf serum (FCS)</td>
			<td>Invitrogen</td>
			<td>10091148</td>
			<td>heat-inactivated (30 min, 56&#176;C)</td>
		</tr>
		<tr>
			<td>filter 0.22&#181;m</td>
			<td>Sarstedt</td>
			<td>83.1826.001</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-coupled anti-mouse secondary antibody</td>
			<td>santa cruz</td>
			<td>sc-2005</td>
			<td>use 1:10,000 in 5% milk powder in TBS-T</td>
		</tr>
		<tr>
			<td>HRP-coupled anti-rabbit secondary antibody</td>
			<td>santa cruz</td>
			<td>sc-2004</td>
			<td>use 1:10,000 in 5% milk powder in TBS-T</td>
		</tr>
		<tr>
			<td>4x Laemmli loading buffer, Roti-Load 1</td>
			<td>Roth</td>
			<td>K929.1</td>
			<td></td>
		</tr>
		<tr>
			<td>microfuge SIGMA 1-15K</td>
			<td>Sigma Laborzentrifugen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>milk powder (Blotting-Grade Blocker, nonfat dry milk)</td>
			<td>BioRad</td>
			<td>170-6404</td>
			<td></td>
		</tr>
		<tr>
			<td>multifuge 3 L-R</td>
			<td>Heraeus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoSight LM10</td>
			<td>NanoSight Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, w/o Ca and Mg</td>
			<td>Pan biotech</td>
			<td>P04-36500</td>
			<td></td>
		</tr>
		<tr>
			<td>perfusor syringe 50 mL</td>
			<td>Braun</td>
			<td>8728844F</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau-S staining solution</td>
			<td>PanReac AppliChem</td>
			<td>A2935,0500</td>
			<td></td>
		</tr>
		<tr>
			<td>rotor Sw32.1 Ti for ultracentrifugation (6x 17 mL)</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>rotor TLA-120.2 for ultracentrifugation (10x 1.5 mL)</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tubes for flow cytometry (5 mL, round-bottom)</td>
			<td>BD Biosciences</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>tubes for ultracentrifugation (15 mL)</td>
			<td>Beckman Coulter</td>
			<td>344061</td>
			<td></td>
		</tr>
		<tr>
			<td>tubes for ultracentrifugation (11* 34 mm)</td>
			<td>Beckman Coulter</td>
			<td>343778</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubulin antibody</td>
			<td>Millipore</td>
			<td>05-829</td>
			<td>1:5000 in 5%BSA in TBST</td>
		</tr>
		<tr>
			<td>ultracentrifuge Optima L-80 XP</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ultracentrifuge TL-100</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>valve filter Seraplas V15</td>
			<td>Sarstedt</td>
			<td>53,428</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of microvesicles from peripheral blood

外囊泡（电动汽车），包括小胞内源性外来体（的Exos，直径&lt;100纳米）和大质膜来源的微泡（MVS，直径&gt; 100纳米）的发布是发生在所有活细胞的基本细胞过程。这些特异于原籍细胞和体外研究囊泡运输蛋白质，脂质和核酸都强调作为细胞间通讯的介质的重要性。电动汽车从各种体液被成功地分离和血液尤其电动汽车已被确定为有前途的癌症或感染性疾病的生物标志物。为了让血液MV亚群的研究，我们提出了从外周血样本的MV的标准化分离和鉴定的协议。的MV从由差速离心EDTA抗凝血浆样品沉淀，并通常具有的直径为100 - 600纳米。由于其较大的尺寸，它们可以容易通过流式细胞术，即经常在临床诊断中使用的和在大多数实验室可用的技术进行研究。为隔离MV的质量控制测定中的几个例子将给出并且可用于在血液不同的MV亚群的鉴别标记物将提交。

为了量化可被分离之后的所述协议的MV的产率，计算从10供体的血液样品中分离出的MV的量。的MV产量，这是在一个洛瑞蛋白质测定评估，范围从10至30微克以每毫升血液19.2微克的MV（ 表1）的平均。由纳米颗粒跟踪分析（NTA）确定颗粒浓度范围为1.66×10 9 2.36×10 10与每mL血浆样品5.9×10 9颗粒（ 表2）的平均值。通过透射电子显微镜的MV的进一步表征显示，是由脂双层包围，并没有包含任何细胞器（ 图1A）的直径&gt; 100nm的囊泡的群体。 NTA确认了分离出的MV的大小从100范围可达600纳米（ 图1B）和平均MV大小为201纳米（ 图1C）。染色典型MV和外切标记通过Western印迹证明了孤立的MV为阳性微管蛋白和仅表现CD9和CD81的轻微表达，而的Exos为阴性微管蛋白和CD9和CD81（ 图2）富集。 通过流式细胞术（ 图3A）的分离的MV的分析表明，可以使用通常用于从细胞培养物上清液中分离的MV相同的参数来选通一个限定囊泡种群。那是由PBS中的测量获得的背景信号明显不同+ 1％的囊泡的贫的FCS不加入的MV（ 图3B）的。为了分析存在于血液中的不同的MV种群的MV用既定标记染色的不同血细胞种群， 例如 ，CD62P对血小板衍生的MV，CD45白细胞衍生的MV，CD235a为红血细胞来源的MV和CD62E内皮细胞衍生的MV（ 图4）。这表征表明MV亚群的不同百分比的调查供体血液样本中，而大多数的MV似乎是由血小板在所有样品中脱落。 <img alt="图1" src="/files/ftp_upload/55057/55057fig1.jpg" /> 图1:从外周血中分离出的MV的大小分布。 A，孤立的MV是由透射电子显微镜观察。 B，MV的示出囊泡的尺寸分布的代表性纳米粒子追踪分析（NTA）。 C，自10个独立的制剂的平均MV尺寸通过NTA（平均）测量。 <a href="http://ecsource.jove.com/files/ftp_upload/55057/55057fig1large.jpg" target="_blank">请点击此处查看人</a>arger版本这个数字。 <img alt="图2" src="/files/ftp_upload/55057/55057fig2.jpg" /> 图2:Western 印迹隔离MV的表征。从两个供体分离的MV和差异的Exos蛋白的表达，用Western印迹可视化。 <a href="http://ecsource.jove.com/files/ftp_upload/55057/55057fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55057/55057fig3.jpg" /> 图3: 流式细胞仪的MV分析。 A，是的MV首次显现远期（FSC）与sidescatter（SSC）阴谋门在各自的MV人口。随后，这些的MV的特征在于对于感兴趣的抗原<html>使用针对抗原的荧光标记的抗体。 B，典型的FSC对从两个供体的血浆中分离出的MV SSC地块。作为比较，对从细胞培养上清液以及阴性对照分离的A549肺癌细胞只用PBS + 1％囊泡贫的FCS而不的MV肿瘤细胞衍生的MV的典型曲线图中示出在右边。 <a href="http://ecsource.jove.com/files/ftp_upload/55057/55057fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55057/55057fig4.jpg" /> 图4: 通过流式细胞仪分离的MV的表征。从三个供体的MV进行表征为建立血细胞标记（红色）通过流式细胞术的表达。各个同种型对照以灰色显示。http://ecsource.jove.com/files/ftp_upload/55057/55057fig4large.jpg"目标="_空白"&gt; 点击此处查看该图的放大版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 样品 </td><td> MVS / mL的血[微克] </td></tr><tr><td> ＃1 </td><td> 29.4 </td></tr><tr><td> ＃2 </td><td> 10.3 </td></tr><tr><td> ＃3 </td><td> 15.2 </td></tr><tr><td> ＃4 </td><td> 31.1 </td></tr><tr><td> ＃5 </td><td> 18.8 </td></tr><tr><td> ＃6 </td><td> 22.7 </td></tr><tr><td> ＃7 </td><td> 19.1 </td></tr><tr><td> ＃8 </td><td> 18.7 </td></tr><tr><td> ＃9 </td><td> 15.0 </td></tr><tr><td> ＃10 </td><td> 11.9 </td></tr></tbody></table> 表1:从外周血MV蛋白质产量的影响。 </str翁&gt;显示的是一个从10供体绘制每毫升外周血MV的量。 MV产量通过Lowry法进行定量。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 样品 </td><td> MVS /毫升血浆[粒子数] </td></tr><tr><td> ＃1 </td><td> 6.42E + 09 </td></tr><tr><td> ＃2 </td><td> 2.36E + 10 </td></tr><tr><td> ＃3 </td><td> 1.88E + 09 </td></tr><tr><td> ＃4 </td><td> 6.51E + 09 </td></tr><tr><td> ＃5 </td><td> 3.48E + 09 </td></tr><tr><td> ＃6 </td><td> 4.57E + 09 </td></tr><tr><td> ＃7 </td><td> 2.09E + 09 </td></tr><tr><td> ＃8 </td><td> 1.66E + 09 </td></tr><tr><td> ＃9 </td><td> 2.54E + 09 </td></tr><tr><td> ＃10 </td><td> 6.20E + 09 </td></tr></tbody></table> 表2:MV粒子收益率从外周血标本。 </st荣&gt; MV是从10个捐助国和粒子计数是由纳米粒子跟踪分析测定血浆样品中分离。示出的是来自三个独立测量的平均值。

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Isolation and Characterization of Microvesicles from Peripheral Blood

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-and-characterization-of-microvesicles-from-peripheral-blood

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

22.9K 次观看.  University Medical Center Göttingen. 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. 

视频: Isolation and Characterization of Microvesicles from Peripheral Blood

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 ...

科研

生物学

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 ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

代人类诱导多能干细胞从外周血中使用STEMCCA慢病毒载体

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

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 ...

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 ...

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin&#39;s lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.

cd4+ t 细胞的分离及6色流式细胞仪分析外周血中循环 t-滤泡 (ctfh) 细胞亚群

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the ...

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 of Human Neutrophils from Whole Blood and Buffy Coats

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells recruited at the site of inflammation via rapid extravasation through vessels. There, neutrophils perform an array of functions to kill invading pathogens and mediate immune signaling. Freshly purified neutrophils from human blood are the model of choice for study, as no cell line fully replicates PMN functions and biology. However, neutrophils are short-lived, terminally differentiated cells and are highly susceptible to activation in response to physical (temperature, centrifugation speed) and biological (endotoxin, chemo- and cytokines) stimuli. Therefore, it is crucial to follow a standardized, reliable, and fast method to obtain pure and non-activated cells. This protocol presents an updated protocol combining density gradient centrifugation, red blood cell (RBC) sedimentation, and RBC lysis to obtain high PMN purity and minimize cell activation. Furthermore, methods to assess neutrophil isolation quality, viability, and purity are also discussed.

This protocol details a method for the isolation of neutrophils from whole blood, buffy coats, or leukapheresis membranes, achieving good yield, high purity, and minimal cell activation. We utilize gradient purification, red blood cell (RBC) sedimentation, and RBC lysis to obtain a high-quality/purity neutrophil preparation.

从全血和巴菲皮脂中分离出人类嗜中性粒细胞

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells ...