Name: isolation and characterization of microvesicles from peripheral blood
Uploaded: 2017-01-06T11:00:00.000+00:00
Duration: 6 min 3 s
Description: Watch this Scientific Journal Video about Isolation and Characterization of Microvesicles from Peripheral Blood at JoVE.com

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><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 H&lt;sub&gt;2&lt;/sub&gt;O and adjust pH to 7.6</td></tr><tr><td colspan="4">For 1x TBS-T mix 100 mL 10x TBS with 900 mL H&lt;sub&gt;2&lt;/sub&gt;O and add 1 mL Tween-20</td></tr><tr><td>CD235a-PE</td><td>Beckman Coulter</td><td>A07792</td><td>use 5 &amp;micro;l for staining</td></tr><tr><td>CD45-FITC</td><td>Beckman Coulter</td><td>7782</td><td>use 5 &amp;micro;l for staining</td></tr><tr><td>CD62E-PE</td><td>Biolegend</td><td>336008</td><td>use 0.8 &amp;micro;g for staining</td></tr><tr><td>CD62P-PE</td><td>Biolegend</td><td>304905</td><td>use 0.1 &amp;micro;g for staining</td></tr><tr><td>CD81 antibody</td><td>Biolegend</td><td>349501</td><td>1:2,000 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>1:1,000 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 &amp;deg;C)</td></tr><tr><td>filter 0.22 &amp;micro;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 (6 x 17 mL)</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>rotor TLA-120.2 for ultracentrifugation (10 x 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>1:5,000 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="https://ecsource-jove-com.bdigitaluss.remotexs.co/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="https://ecsource-jove-com.bdigitaluss.remotexs.co/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="https://ecsource-jove-com.bdigitaluss.remotexs.co/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。https://ecsource-jove-com.bdigitaluss.remotexs.co/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。

Watch this Scientific Journal Video about Isolation and Characterization of Microvesicles from Peripheral Blood at JoVE.com

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 San Sebastian

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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

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released from many cell types and play a pivotal role in regulating cell-cell communication and a diverse range of biological processes. Many different methods for the characterization of EVs have been described. However, most of these methods have the disadvantage that the preparation and characterization of the samples are very time-consuming, or it is extremely difficult to analyze specific markers of interest due to their small size and due to the lack of discrete populations. While methods for analysis of EVs have been considerably improved over the last decade, there is still no standardized method for characterization of single EVs. Here, we demonstrate a semi-automated method for characterization of single EVs by fluorescence-based nanoparticle-tracking analysis. The protocol that is presented addresses the common problem of many researchers in this field and provides the complete workflow for rapid isolation of EVs and characterization with PKH67, a general cell membrane linker, as well as with specific surface markers such as CD63, CD9, vimentin, and lysosomal-associated membrane protein 1 (LAMP-1). The presented results show a high level of reproducibility, as confirmed by other methods, such as Western blotting. In the conducted experiments, we exclusively used EVs isolated from human serum samples, but this method is also suitable for plasma or other body fluids and can be adjusted for characterization of EVs from cell culture supernatants. Irrespective of the future progress of research on EV biology, the protocol that is presented here provides a rapid and reliable method for rapid characterization of single EVs with specific markers.

In this protocol, we describe the complete workflow for rapid isolation of extracellular vesicles from human whole blood and characterization of specific markers by fluorescence-based nanoparticle-tracking analysis. The presented results show a high level of reproducibility and can be adjusted to cell culture supernatants.

基于荧光的人体血液单细胞外囊快速表征

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

科研

生物化学

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides (MF) and S&#233;zary syndrome (SS) represent the most common subtypes. Despite a number of studies on characterizing gene expression, genetic alterations, and epigenetic abnormalities of CTCL, the molecular pathogenesis of MF/SS remains unclear. MF refers to the more common CTCL with a skin-predominance, and is usually limited to skin, whereas&#160;SS is an aggressive leukemic variant of CTCL with widespread skin involvement and is characterized by neoplastic distribution mainly involving blood, skin, and lymph node. Focusing on clinical practice, the identification of gene expression biomarkers has&#160;enormous potential to improve diagnosis and treatment of MF/SS. Indeed, recent transcriptomic studies have identified potential diagnostic biomarkers from differences in gene expression between normal and malignant T cells, which may improve our understanding of SS biology, and reveal potential therapeutic targets. This manuscript describes a detailed reproducible protocol for the isolation of peripheral blood mononuclear cells from fresh whole blood from patients diagnosed with SS, selection of CD4+ memory T cells (CD4+CD45RO+ T cells), chemical stimulation, and preparation of RNA suitable for transcriptomic profiling to discover novel prognostic molecular markers to gain additional insight in disease etiology. The stimulation using chemical agonist to activate nuclear regulation provides more specific assessment for pathways important in the dynamic transcription regulation and gene expression and eliminates confounding defects that may arise from upstream signaling defects arising from TCR antigen loss at the cell membrane. The data obtained from comparison of transcriptome of unstimulated to stimulated SS T cells unmasks functional regulatory gene expression defects not evident from analysis of quiescent unstimulated cells. Furthermore, the method outlined from this approach can be adapted for studying T cell gene expression defects in other T cell immune diseases.

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with S&#233;zary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

从Sézary综合征患者中分离人外周血单核细胞和CD4 + T细胞以进行转录组学分析

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

癌症研究

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 &#956;l. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease.

This protocol details a method to isolate extracellular vesicles (EVs), small membranous particles released from cells, from as little as 10 &#956;l serum samples. This approach circumvents the need for ultracentrifugation, requires only a few minutes of assay time, and enables the isolation of EVs from samples of limited volumes.

基于纸张的设备进行隔离外囊泡与表征

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

生物工程

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

从HIV-1阳性者的血浆分离外来体的

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

免疫与感染

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

分离和剖析人力胆汁含外来体的MicroRNA-

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

医学

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) containing immunologically active molecules. Knowledge regarding the molecular mechanisms of vesicle biogenesis, the content of the vesicles, and their functions at the pathogen-host interface is very limited. Addressing these questions requires rigorous procedures for isolation, purification, and validation of EVs. Previously, vesicle production was found to be enhanced when M. tuberculosis was exposed to iron restriction, a condition encountered by Mtb in the host environment. Presented here is a complete and detailed protocol to isolate and purify EVs from iron-deficient mycobacteria. Quantitative and qualitative methods are applied to validate purified EVs.

Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

铁限分枝杆菌产生的细胞外囊泡的分离和特征

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) ...

Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular effectors. In rats, spleen MVs (SMVs) are surrogate markers of pathophysiological conditions. Previous in vitro studies demonstrated that Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, enables&#160;the endothelial shedding and apoptosis while lipopolysaccharide (LPS) favors the shedding of splenocyte-derived microvesicles (SMVs). In vivo studies showed the feasibility of pharmacological control of SMV shedding. The present protocol establishes a standardized procedure for isolating splenic SMVs from the P. gingivalis acute murine infection model.&#160;P. gingivalis infection was induced in young C57BL/6 mice by intraperitoneal injection (three injections of 5 x 107 bacteria/week). After two weeks, the spleens were collected, weighed, and the splenocytes were counted. SMVs were isolated and quantified by protein, RNA, and prothrombinase assays. Cell viability was assessed by either propidium iodide or trypan blue exclusion dyes. Following splenocyte extraction, neutrophil counts were obtained by flow cytometry after 24 h of splenocyte culture. In P. gingivalis-injected mice, a 2.5-fold increase in spleen weight and a 2.3-fold rise in the splenocyte count were observed, while the neutrophils count was enhanced by 40-folds. The cell viability of splenocytes from P. gingivalis-injected mice ranged from 75%-96% and was decreased by 50% after 24 h of culture without any significant difference compared to unexposed controls. However, splenocytes from injected mice shed higher amounts of MVs by prothrombinase assay or protein measurements. The data demonstrate that the procoagulant SMVs are reliable tools to assess an early spleen response to intraperitoneal&#160;P. gingivalis infection.

Microvesicles shed from the plasma membrane act as cellular effectors. Spleen microvesicles (SMVs) are surrogate markers of pathophysiological conditions. In rats and mice, SMV content and properties characterize aging or inflammation and are altered by cytoprotective treatments, making them a valuable yet abundant monitoring tool for preclinical research.

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular ...

生物学

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

从血浆和实体组织中分离和分析可追溯和功能化的细胞外囊泡

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

培养的人间充质干细胞细胞外囊泡的分离、表征和治疗应用

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

<meta charset="utf8"></head><body>使用血浆细胞外囊泡蛋白质组的心血管生物标志物研究

Isolation, Characterization, and Proteomic Analysis of Plasma-Derived Extracellular Vesicles for Cardiovascular Biomarker Discovery

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research due to their role in regulating intercellular communication, potentially serving as valuable biomarkers for cardiovascular disease. However, the EV proteome and its potential as a biomarker in cardiovascular diagnostics remain poorly understood. This protocol presents a standardized method for the isolation and quantification of plasma-derived EV and the analysis of their protein cargo using plasma samples from patients presenting to the Chest Pain Unit of a large university hospital. Following routine phlebotomy, EV are isolated from plasma by differential ultracentrifugation. The enrichment of specific EV marker proteins in EV isolates is visualized by immunoblotting, and average size distribution and plasma EV concentrations are quantified by nanoparticle tracking analysis. Finally, ultra-performance liquid chromatography-tandem mass spectrometry is employed for label-free analysis of the EV proteome. This protocol thus provides a comprehensive approach to study and use plasma-derived EV as potential carriers of critical biological information as well as to explore their potential as novel biomarkers.

<meta charset="utf8"></head><body>作者聚焦：推进血浆细胞外囊泡蛋白质组分析用于心血管生物标志物研究

This article describes a methodology for the isolation, characterization, and quantification of human plasma-derived extracellular vesicles (EV) and presents a workflow for label-free analysis of the EV proteome using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

血浆来源的细胞外囊泡的分离、表征和蛋白质组学分析用于心血管生物标志物的发现

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research ...