笔者想感谢戴尔Hirschkorn从血液系统研究所对他的帮助与流式细胞仪的仪器设置。这项工作是由美国国立卫生研究院支持授予HL095470和U01 HL072268和国防部合同W81XWH-10-1-0023和W81XWH-2-0028。

注:以下协议已符合已执行与人类福祉所有机构，国家和国际准则。所有的人的主体样本下机构审查委员会（IRB）批准的协议，并与主体的知情同意测试。 1.方法A:个别检测方法电动汽车的血液采样/隔离1.1）处理<ol><li>抽取血液从供血者/病人到使用以下2步差速离心协议含1.5毫升ACD-A液等立即适合抗凝和过程（在30分钟内最多）两个10毫升玻璃试管。 注意:此协议将从合并〜17毫升的抽血收率约贫血小板血浆（PPP）的10毫升如果有更多的或更少的PPP是需要的，血液收集管的数量可以相应地调整。 </li><li>离心样品在1500×g离心在室温10分钟到等离子体从血沉棕黄层和红细胞中分离出来。转让1.2毫升等分血浆上清至1.5 ml离心管，小心不要打扰含棕黄色外套和红细胞的底层。 </li><li>旋转以13,000×g离心10分钟，在室温以除去血小板和大细胞碎片。小心转移PPP，留下200微升，以避免干扰颗粒，并添加在PPP到一个新的试管中。 </li><li>在这一点上，立即使用PPP用于分析或于1.0ml等份转移至新的1.5毫升离心管中并储存在-80℃下长达两年以后的分析（参见图1A，用于总览）。 </li><li>如果需要纯化的电动车进行功能试验，转印6毫升PPP协议到超速离心管中，并添加28的0.2微米过滤的磷酸盐缓冲盐水液（PBS）中。自旋用于使用配备吊桶式转子超速离心机以100,000×g离心60分钟，在RT。吸上清，重悬EV像素让在1.5ml培养基。 注:为获得最高的重现性，血液样品应尽可能一致处理来自捐助者捐助。任何变化的EV隔离方法能显著影响检测电动车辆的数量和类型。 </li></ol> 1.2），准备样品进行分析注意:从这个角度上，该步骤说明高通量协议分析12个样品14标记的3板。然而，在这里可以使用的抗体的其它组合;该协议可以适于通过为那些感兴趣代建议的标记物来研究其它的EV种群。 <ol><li>从冰箱中取出12个样品（如果储存在-80℃）和解冻，在37℃。 </li><li>吸取内容上下几次混合。从每个样品中取出320微升，并加入到96孔板的顶行。 注:宽度可调多井吸管是这和整个屁股许多其他步骤非常有帮助唉，分析多个样品尤其是当一次。 </li></ol> 1.3）染色EV样品<ol><li>染色前，将过滤所有抗体（ABS）以除去聚集物，这可能会导致阳性信号。 <ol><li>要使用在各3片的结合的抗体成使用固定角度单速离心机（〜750×g离心）在RT 2分钟分开0.22μm的离心过滤管和离心机，或直到所有的抗体混合物通过过滤器已通过和无抗体的液体保留在过滤器的表面上。每次使用前店抗体鸡尾酒在冰箱里长达两个星期，但再次过滤器。 </li></ol></li><li>加过滤抗体混合物的合适量，以每孔在第2行（ 例如 ，在面板样品I的染色的2微升每Ab的，所以共有12微升过滤抗体鸡尾酒的是，每孔加入到行2）。请参考图1B为建议的板图的轮廓。重复这些除s至下方，如果更多的面板上运行的行（在此，加入8微升/孔的第二小组鸡尾酒到行3和11微升/孔的组III鸡尾酒至4行的是指材料清单用于特定面板信息）。 </li><li>使用多通道移液管，混合在PPP样品中第1行上下和从孔中第1行中的行转移100μl到井2.混合上下。变化的提示和重复，转移100μl的从第1行至行3和4孵育在4℃下进行30分钟。 </li></ol> 1.4）洗涤MV样本<ol><li>除去96孔板在4℃，并转移至生物安全柜中。使用多通道移液管，添加220微升PBS /孔的行6-8（用于冲洗/清洗含有染色PPP协议的孔）。 </li><li>传输的每一个的内容以及使用宽度可调多管移液器（对于12个样本的预标记的离心过滤管，用3片的抗体，12×3 = 36过滤管将需要）。利用同样的技巧，从洗行取出200微升PBS，并添加到从PPP只是删除了相应的井。 </li><li>混合上下冲洗孔和冲洗液转移至向其中PPP的先前溶液中加入相同的过滤器。关闭顶部离心式过滤器。变化的提示。 </li><li>重复此过程，与剩余的染色样品，直到所有的染色样品的PPP已经转移连同它们漂洗溶液至离心过滤器。 </li><li>转移离心过滤器以一个固定的转子离心机和旋转，在850×g离心3分钟，在RT。 注:确保没有液体留在过滤器顶部。离心后，将过滤器应显示为"干"的，顶部没有可见的流体层残留。而不太可能的，一定的PPP样品可能需要更长的离心时间，以有效地移动穿过所述过滤器。 </li><li>取下离心过滤管，并返回到生物安全柜中。使用多通道移液管，重悬过滤器的顶部在300微升PBS中。转移悬浮内容预先贴标签的试管立即FCM分析。 注意:要保持吸液管柱塞凹陷样品间一致的力和数量，以避免样品到样品的变化是非常重要的。这最好应使用电子移液器已编程到吸管上下一个特定体积进行（ 例如 ，280微升）的每个采样时间（ 例如 ，8倍）的具体数量。 </li></ol> 1.5），流式细胞仪设置<ol><li>打开FCM软件。在此之前实验设置，使用的仪器设置珠（以下制造商的说明）进行日常仪器校准和设置。 </li><li>如果EV样本已染色与多于一个的抗体和多个荧光染料都可以一次进行测量，计算出的补偿值如下: <ol><li>加入2滴赔偿珠预标记的管（1管为每个荧光染料缀合的抗体）以及添加抗体的推荐量。添加2滴负补偿珠到另一个管作为未染色补偿控制来使用。 </li><li>孵育在4℃下进行30分钟，用PBS重悬洗于400μlPBS中。 </li><li>使用流式细胞仪的软件流程包括与仪器，运行每个补偿管荧光灯调整电压，将每个峰值约10 4 5数十年来规模。确保该荧光峰是最高（最亮）在其自己的信道的荧光相比，所有其它信道，并在必要时再调整的荧光参数的电压。运行每个单独染色补偿管，捕捉每管至少有5,000个事件。 </li><li>选择标签"实验"，然后选择"补偿"，然后选择"计算赔偿"申请补偿值的所有样本。 </li></ol></li><li&gt;设置前向散射（FSC）和侧向散射（SSC）的电压参数，以对数比例，并选择由流式细胞仪（FSC = 200和SSC = 200）为每个所允许的最低阈值。 </li><li>在运行的0.22微米过滤的PBS中的管，调整FSC和SSC电压到该排除多数的背景噪声（ 即 ，恰好低于该事件的发生率超过5个事件/秒的电压阈值）的最高值。 </li><li>接下来，运行含有0.2微米的管 - 1.0微米的珠子稀释在PBS中，如果必要的。在一个FCS与SSC的情节，画周围的珠子群门之间0.2微米和1.0微米捕获事件。可替代地，在一个SSC-H的直方图，画出一个门，以包括所有的事件比1.0微米小球要小。 </li><li>流式细胞仪的流速为"Lo"（约8-12微升/分钟）。使用珠管（或含有珠已知浓度等管）调整流式细胞仪流速盘，直到前夕NT率达到约200次/秒。读取所有样品管在相同的流速，并使用在以后的实验中相同的珠浓度，以确保流速保持运行之间是一致的。 </li><li>运行在PBS中稀释彩虹荧光发色粒子的管。获得5000事件。记录对FSC，SSC和每个颜色通道的平均强度值。使用这些值来调整在以后的实验中的电压，以确保荧光强度保持实验之间保持一致。 </li></ol> 1.6）样品阅读<ol><li>流式细胞仪的流动速率设定为"Lo"的（约8-12微升/分钟）和运行每个样品完全1或2分钟。 </li><li>第一读取后，10％的加20微升NP-40的每个样品，移液器上下，并重新读取为相同量的时间（1或2分钟），以允许在检测到的阳性事件的减法在相同的时间内裂解样本。 注:这是非常小鬼ortant该样品用吸管，而不是涡流混合。根据我们的经验，涡旋可能会导致一些抗体的自聚集，导致EV-模仿阳性事件。 </li><li>一旦所有样品已阅读，出口用于使用FCM分析软件进一步分析所有.fcs文件到一个单独的文件。 </li></ol> 1.7）数据分析<ol><li>打开FCM分析软件。导入所有的.fcs文件到一个新的实验文件。 </li><li>打开珠只管。在FSC-A对SSC-A地块，周围画一个栅极之间0.2微米和1.0微米大小的所有珠子。这是EV栅极。拖动来添加到所有样品。 </li><li>使用裂解的样品作为阴性对照，绘制在门的背景荧光中使用的各荧光通道的边缘。拖动荧光门到每个相应的未裂解的样品的EV栅极。 注:在这一点上也很有研究双荧光双参数图。火箭形状双阳性象限（ 见图8），特别是如果标记物是已知的，以驻留在无关的细胞类型，可指示由聚集或其它囊泡-模拟事件的假象。 </li><li>对于每一个荧光标记物，减去从非裂解样品中的事件数的裂解样品中的事件数。任选地，由电动车辆的总数除以该号码非裂解的EV栅内以获得％正值。 </li></ol> 2.方法B:磁珠法电动汽车的血液采样/隔离2.1）处理<ol><li>请参阅方法A（1.1节）中描述的血液处理方法。 </li></ol> 2.2），准备样品进行分析<ol><li>如果需要的话，分馏PPP或超速离心电动车到外来体和微泡。加入250微升PPP或超离心电动车到0.22微米的离心过滤器，并转移到一个固定的转子离心和旋转750 Xg下在室温下2分钟。 注:确保没有液体留在过滤器顶部。离心后，将过滤器应显示为"干"的，顶部没有可见的流体层残留。而不太可能，某些样品，可能需要稍长的离心时间为流体有效地移动穿过所述过滤器。 </li><li>在2ml洗未涂覆6微米的聚苯乙烯小珠（ 例如 ，负ABC珠）用2X RPMI培养基，重悬。加入6000珠子每个FACS管。阴性对照管中，单独添加400微升RPMI培养基到珠。所有其他管，加入200μlPPP或电动车超速离心（或其部分）和200微升RPMI媒体。 </li><li>调整所有管的终体积为400微升用培养基孵育过​​夜，在4℃在振荡器上。 </li><li>第二天早上，洗珠用2ml培养基。吸脱上清。 </li><li>用5％的牛血清白蛋白（BSA）的培养基（400微升）处理3小时，在4方框＃176; C对振动筛。 </li><li>用2ml培养基洗涤珠。吸并重新悬浮颗粒在100微升介质。 </li></ol> 2.3）染色EV样品<ol><li>过滤所有的抗体。使用方法A中使用的相同的抗体的面板，或者如果需要的话，只要它们的荧光染料是彼此相容创建抗体的不同组合。 </li><li>要使用在单个面板结合的所有抗体为0.22μm的离心过滤管。离心机利用固定角单速离心2分钟，或直至所有的抗体混合物的已通过过滤器和无抗体的液体保留在过滤器的表面上。 </li><li>添加过滤抗体混合物适当的音量，以所有管和孵育30分钟，在4℃。 </li><li>用2ml的培养基，重悬在400微升的媒体上流式细胞仪洗珠并立即运行（或在同一天之内）。 </li></ol> 2.4），流式细胞仪设置和样品阅读<ol><li>打开FCM软件。在此之前实验设置，使用的仪器设置珠（以下制造商的说明）进行日常仪器校准和设置。 </li><li>如果EV样本已染色与多于一个的抗体和多个荧光染料都可以一次进行测量，计算出的补偿值如下: <ol><li>加入2滴赔偿珠预先标记的管（1管各荧光标记的抗体），并添加抗体的推荐量。添加2滴负补偿珠到另一个管作为未染色补偿控制来使用。孵育在4℃下进行30分钟，用PBS重悬洗于400μlPBS中。 </li><li>用FCM软件，运行每个补偿管荧光灯调整电压将每个峰值约10 4 5数十年来规模。确保该荧光峰是最高（最亮）在其自己的信道的荧光相比，所有其它信道，并如果需要再次调整荧光参数电压。运行每个单独染色补偿管，捕捉每管至少有5,000个事件。 </li><li>选择标签"实验"，然后"补偿"，然后"计算赔偿"申请补偿值的所有样本。 </li></ol></li><li>更改FSC和SSC电压参数，登录规模和选择每个通过流式细胞仪（FSC = 200和SSC = 200）所允许的最低门槛。 </li><li>运行样品，门上的单珠人口和收购这个门2000的事件。出口.fcs文件。 </li><li>用FCM分析软件分析.fcs文件。门上的单珠。计算几何平​​均荧光强度（MFI）针对每个荧光染料和与阴性对照的MFI进行比较。 </li></ol>

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作者有没有利益冲突披露。

为隔离，治疗和电动汽车的分析两种不同的协议采用两种单独的检测或珠为基础的方式呈现。选择最适当的方法来使用并不总是直截了当，需要样品的理解被测试以及感兴趣的个体的亚群。而且，用于获取细胞仪的灵敏度，必须在选择最合适的方法可以考虑。通常情况下有使用的，而是相结合的方法提供了比单独使用任何一种方法的示例的详细信息没有一个最佳的方案。理想情况下，几种不同的?...

胞外囊泡（电动汽车）的体液中是高度地参与细胞 - 细胞通讯而有助于调节生物过程的一个不同的范围中找到的小，膜衍生的囊泡。使用流式细胞仪（FCM）电动车的分析已经非常困难，由于其体积小，缺乏积极的兴趣标记离散人口。方法EV分析，而在过去十年显着改善，仍处于进展中的工作。不幸的是，没有任何一个尺寸适合所有的协议，并确定最适当的方法使用时的几个方面必须加以考虑。这里介绍几种不同的技术来处理电动车和两个协议使用两种独立检测或珠为基础的方法分析了电动车。在一个合理的˚F这里描述将协助消除抗体聚集体在商业制剂中常见的方法中，增加信噪比，以及设置门ashion最小化检测的背景荧光。第一协议使用一个单独的检测方法，该方法是特别适合用于分析大量的临床样品中，而第二协议使用基于珠子的方式来捕获和检测小电动汽车和外来体。 电动汽车，也被称为微粒，是在参与细胞-细胞通讯而这有助于调节生物过程1的各种各样的体液发现小，膜衍生的囊泡。通过各种表面标记物和/或生物材料直接转移表达，电动汽车能够改变受体细胞的功能发挥要么激活或抑制细胞间通讯2角色- 4。在临床上，血小板衍生的电动汽车已知具有强抗凝血活性5，而另一些已被证明有助于广泛的条件下，从促进肿瘤metasta矽统6〜防止疾病7。电动车可分为较小类别的细胞衍生囊泡诸如外来体和微泡（MVS），这取决于它们的尺寸和产生8的机制。细胞衍生的小泡的亚群的命名法继续进行的辩论8,9的话题，但是，外来体通常被描述为小，来自内体的融合与质膜派生40至100纳米的颗粒，而MV的是大100〜1,000纳米粒子脱落质膜10的形成。这里，一般的术语"电动汽车"将被用来指所有类型的细胞外生物囊泡由细胞释放。 从全血中电动车辆的隔离是一个多步骤的过程和许多不同的处理的变量已被证明影响的EV内容，包括贮存温度和持续时间11,12，抗凝血剂/防腐剂使用13和离心法使用14。有必要对这些变量的标准化，导致血栓与止血科学和标准化委员会（SSC ISTH）进行正常的血液处理和EV隔离程序15,16由国际协会的建议，但存在着研究人员之间的最优协议没有达成共识使用12。然而，大多数人认为，这严格控制分析前变量是准确的和可重复的数据是至关重要的。 为了分析电动汽车中，研究人员已利用各种方法，包括透射电子显微镜17，扫描电镜18,19，原子力显微镜，动态光散射20,21和蛋白质印迹22,23。而FCM是选择用于许多研究者9,24的方法- 26由于其高吞吐量能力，使用流式细胞仪电动车辆的分析已经32 -由于其规模和缺乏独立阳性群体27个老大难问题。相比细胞的分析，对电动汽车的结果1）较少的荧光由于每个颗粒抗原的数量越少发射的小尺寸和后染色洗涤2）有限的可行性，这是必要的，以减少背景荧光。研究人员共同面临的挑战包括免疫球蛋白聚集27,28和抗体29的自聚集所产生的信号。此外，较长的处理时间和冗长的洗涤/分离过程中使用的许多当前的协议33,34需要多天时间承诺来分析少量的样品，使它们小于适用于高通量应用。一些研究人员干脆放弃洗涤步骤，使传统上使用的FCM阴性对照，如荧光减一（FMO）及抗体亚型无用的准确评估BACkground荧光30。 我们的协议处理，可以阻碍电动车的正常的FCM分析三个共同的问题:从抗体的聚集体和其它非囊泡，难以除去未结合的抗体，并且缺乏明显的阳性群体所产生的信号。此处所描述的技术将帮助消除抗体聚集体在商业制剂中常见的，增加信噪比，并以合理的方式，最大限度地减少检测的背景荧光设置栅极。两种不同的检测方法是在这里提出:在第一协议使用的各检测方法，该方法是特别适合用于分析大量的临床样品中，而第二协议使用珠为基础的方法来捕获和检测小电动汽车和外来体。

<table><tbody><tr><td>LSR II benchtop flow cytometer</td><td>BD Biosciences</td><td></td><td>3-laser (20 mW Coherent Sapphire 488 nm blue, 25 mW Coherent Vioflame 405 nm violet, and 17 mW JDS Uniphase HeNe 633 nm red)</td></tr><tr><td>FACS Diva software&amp;nbsp;</td><td>BD Biosciences</td><td></td><td>PC version 6.0</td></tr><tr><td>FlowJo software&amp;nbsp;</td><td>Treestar US</td><td></td><td>Mac version 9.6.1 or PC version 7.6.5</td></tr><tr><td>Sphero Rainbow fluorescent particles</td><td>BD Biosciences</td><td>556298</td><td>used to adjust all channel voltages to maintain fluorescence intensity consistency&amp;nbsp;</td></tr><tr><td>Ultra Rainbow fluorescent particles&amp;nbsp;</td><td>Spherotech</td><td>URFP-10-5</td><td>used in addition to Megamix-Plus SSC beads to ensure EV gating consistency from batch to batch</td></tr><tr><td>Megmix-Plus SSC beads</td><td>Biocytex</td><td>7803</td><td>used to adjust FSC and SSC&amp;nbsp; voltages to maintain&amp;nbsp; consistency&amp;nbsp; between runs. Can also used to monitor flow rate and ajust flow rate dial in order to ensure that same flow rate is used in all runs</td></tr><tr><td>AbC Anti-Mouse Bead Kit</td><td>Life Technologies</td><td>A-10344</td><td>used for compensation controls &amp;amp; negative AbC beads used for beads-based method</td></tr><tr><td>Nonidet P-40 Alternative (NP-40) (CAS 9016-45-9)</td><td>Santa Cruz&amp;nbsp;</td><td>sc-281108</td><td>used in the individual detection method only to lyse samples after initial reading for use as negative controls. Stock may be diluted to 1:10 in PBS and stored in fridge for up to 1 month.</td></tr><tr><td>BD TruCOUNT Tubes</td><td>BD Biosciences</td><td>340334</td><td>used whenver absolute EV concentrations are needed</td></tr><tr><td>Ultrafree-MC, GV 0.22 &amp;micro;m Centrifugal Filter Units</td><td>Millipore&amp;nbsp;</td><td>UFC30GVNB</td><td>used to post-stain wash Evs and/or fractionate EVs based on size</td></tr><tr><td>Vacutainer glass whole blood tubes ACD-A</td><td>BD Biosciences</td><td>364606</td><td></td></tr><tr><td>Facs tubes 12x75 polystrene</td><td>BD Biosciences</td><td>352058</td><td></td></tr><tr><td>50 ml Reservoirs individually wrapped&amp;nbsp;</td><td>Phenix</td><td>RR-50-1s</td><td></td></tr><tr><td>Green-Pak pipet tips - 10 &amp;micro;l</td><td>Rainin</td><td>GP-L10S</td><td></td></tr><tr><td>Green-Pak pipet tips -200 &amp;micro;l&amp;nbsp;</td><td>Rainin</td><td>GP-L250S</td><td></td></tr><tr><td>Green -Pak pipet tips - 1,000 &amp;micro;l&amp;nbsp;</td><td>Rainin</td><td>GP-L1000S</td><td></td></tr><tr><td>Stable Stack L300 tips presterilized</td><td>Rainin</td><td>SS-L300S</td><td></td></tr><tr><td>Pipet-Lite&amp;nbsp;XLS 8 Channel LTS&amp;nbsp;Adjustable&amp;nbsp;Spacer&amp;nbsp;</td><td>Rainin</td><td>LA8-300XLS</td><td></td></tr><tr><td>96 well tissue culture plates</td><td>E&amp;amp;K Scientific</td><td>EK-20180</td><td></td></tr><tr><td>RPMI 1640 Media (without Hepes)</td><td>UCSF Cell Culture Facility</td><td>CCFAE001</td><td>media used for bead-based detection method</td></tr><tr><td>Dulbeccos PBS D-PBS, CaMg-free, 0.2 &amp;micro;m filtered</td><td>UCSF Cell Culture Facility</td><td>CCFAL003</td><td></td></tr><tr><td>Ultracentrifuge Tube, Thinwall, Ultra-Clear</td><td>BECKMAN COULTER INC</td><td>344058</td><td></td></tr><tr><td>PANEL I</td><td></td><td></td><td></td></tr><tr><td>CD3 PerCP-Cy5.5</td><td>Biolegend</td><td>344808</td><td>2 &amp;micro;l</td></tr><tr><td>CD14 APC-Cy7</td><td>Biolegend</td><td>301820</td><td>2 &amp;micro;l</td></tr><tr><td>CD16 V450</td><td>BD Biosciences</td><td>560474</td><td>2 &amp;micro;l</td></tr><tr><td>CD28 FITC</td><td>biolegend</td><td>302906</td><td>2 &amp;micro;l</td></tr><tr><td>CD152 APC</td><td>BD Biosciences</td><td>555855</td><td>2 &amp;micro;l</td></tr><tr><td>CD19 A700</td><td>Biolegend</td><td>302226</td><td>2 &amp;micro;l</td></tr><tr><td>PANEL II</td><td></td><td></td><td></td></tr><tr><td>CD41a PerCP-Cy5.5</td><td>BD Biosciences</td><td>340930</td><td>2 &amp;micro;l</td></tr><tr><td>CD62L APC</td><td>Biolegend</td><td>304810</td><td>2 &amp;micro;l</td></tr><tr><td>CD108 PE&amp;nbsp;</td><td>BD Biosciences</td><td>552830</td><td>2 &amp;micro;l</td></tr><tr><td>CD235a FITC</td><td>biolegend</td><td>349104</td><td>2 &amp;micro;l</td></tr><tr><td>PANEL III</td><td></td><td></td><td></td></tr><tr><td>CD11b PE-Cy7</td><td>Biolegend</td><td>301322</td><td>2 &amp;micro;l</td></tr><tr><td>CD62p APC</td><td>Biolegend</td><td>304910</td><td>2 &amp;micro;l</td></tr><tr><td>CD66b PE&amp;nbsp;</td><td>Biolegend</td><td>305106</td><td>2 &amp;micro;l</td></tr><tr><td>CD15 FITC</td><td>exalpha</td><td>X1496M</td><td>5 &amp;micro;l</td></tr><tr><td>CD9 PE</td><td>Biolegend</td><td>555372</td><td></td></tr><tr><td>CD63 APC</td><td>Biolegend</td><td>353008</td><td></td></tr><tr><td>APC-Cy7 Ms IgG2a, &amp;kappa;</td><td>Biolegend</td><td>400230</td><td></td></tr></tbody></table>

techniques for the analysis of extracellular vesicles using flow cytometry

胞外囊泡（电动汽车）的体液中是高度地参与细胞 - 细胞通讯而有助于调节生物过程的一个不同的范围中找到的小，膜衍生的囊泡。使用流式细胞仪（FCM）电动车的分析已经非常困难，由于其体积小，缺乏积极的兴趣标记离散人口。方法EV分析，而在过去十年显着改善，仍处于进展中的工作。不幸的是，没有任何一个尺寸适合所有的协议，并确定最适当的方法使用时的几个方面必须加以考虑。这里介绍几种不同的技术来处理电动车和两个协议使用两种独立检测或珠为基础的方法分析了电动车。在一个合理的˚F这里描述将协助消除抗体聚集体在商业制剂中常见的方法中，增加信噪比，以及设置门ashion最小化检测的背景荧光。第一协议使用一个单独的检测方法，该方法是特别适合用于分析大量的临床样品中，而第二协议使用基于珠子的方式来捕获和检测小电动汽车和外来体。

<html> 图1概述了用于分离和检测用任一胎圈基法或个体的检测方法电动车辆的整体的处理方案。个别检测用流式细胞仪可以很好地用于分析较大的电动车，但大多数流式细胞仪不能够单独检测颗粒小到外来体电动车。甲珠为基础的方法，能够检测小电动汽车，但是，也有使用这种方法相关联的缺点，如表1所述。通常，使用超速离心（有或没有加入蔗糖梯度分级分离过程的...

Watch this Scientific Journal Video about Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry at JoVE.com

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

使用流式细胞仪（FCM）外囊泡（电动车）的测量许多不同的方法存在。确定最合适的方法时使用的几个方面应予以考虑。两个协议用于测量电动车使用的是单独的检测或珠为基础的方法介绍。

对于外小泡的使用流式细胞仪分析技术

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

techniques-for-analysis-extracellular-vesicles-using-flow

Research

JoVE Journal

Biology

23.1K 次观看.  Blood Systems Research Institute. 使用流式细胞仪（FCM）外囊泡（电动车）的测量许多不同的方法存在。确定最合适的方法时使用的几个方面应予以考虑。两个协议用于测量电动车使用的是单独的检测或珠为基础的方法介绍。 

视频: Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

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

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 &#181;m nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.

小细胞外囊泡高通量分离纯化的流式细胞术分选参数优化

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

流量Virometry来分析病毒颗粒的抗原谱和外囊泡

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

免疫与感染

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

细胞调理介质细胞外囊泡的流式细胞学分析

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

生物学

Size Determination and Phenotypic Analysis of Urinary Extracellular Vesicles using Flow Cytometry

Extracellular vesicles, EVs, are a heterogeneous complex of lipidic membranes, secreted by any cell type, in any fluid such as urine. EVs can be of different sizes ranging from 40-100 nm in diameter such as in exosomes to 100-1000 nm in microvesicles. They can also contain different molecules that can be used as biomarkers for the prognosis and diagnosis of many diseases. Many techniques have been developed to characterize these vesicles. One of these is flow cytometry. However, there are no existing reports to show how to quantify the concentration of EVs and differentiate them by size, along with biomarker detection. This work aims to describe a procedure for the isolation, quantification, and phenotypification of urinary extracellular vesicles, uEVs, using a conventional cytometer for the analysis without any modification to its configuration. The method&#39;s limitations include staining a maximum of four different biomarkers per sample. The method is also limited by the amount of EVs available in the sample. Despite these limitations, with this protocol and its subsequent analysis, we can obtain more information on the enrichment of EVs markers and the abundance of these vesicles present in urine samples, in diseases involving kidney and brain damage.

<meta charset="utf8"></head><body>使用流式细胞术测定尿细胞外囊泡的大小和表型分析

This protocol describes a method for the isolation of urinary extracellular vesicles, uEVs, from healthy human donors and their phenotypic characterization by the size and surface marker expression using flow cytometry.

使用流式细胞术测定尿细胞外囊泡的大小和表型分析

Extracellular vesicles, EVs, are a heterogeneous complex of lipidic membranes, secreted by any cell type, in any fluid such as urine. EVs can be of ...

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter.
Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels.
Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.

The latest generation of EV characterization tools are capable of single EV analysis across multiple parameters simultaneously. Nano-flow cytometry measures all biological particles larger than 45 nm without labeling and identifies specific characteristics of subpopulations by a variety of fluorescent labeling techniques.

通过纳流式细胞术表征单个细胞外囊泡跨膜蛋白

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and ...

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 ...

用于了解输出机制的流式细胞术实验方案

Tracking miRNA Release into Extracellular Vesicles using Flow Cytometry

Extracellular vesicles (EVs) are important mediators of cellular communication that are secreted by a variety of different cells. These EVs shuttle bioactive molecules, including proteins, lipids, and nucleic acids (DNA, mRNAs, microRNAs, and other noncoding RNAs), from one cell to another, leading to phenotypic consequences in the recipient cells. Of all the various EV cargo, microRNAs (miRNAs) have garnered a great deal of attention for their role in shaping the microenvironment and in educating recipient cells because of their clear dysregulation and abundance in EVs. Additional data indicates that many miRNAs are actively loaded into EVs. Despite this clear evidence, research on the dynamics of export and mechanisms of miRNA sorting is limited. Here, we provide a protocol using flow cytometry analysis of EV-miRNA that can be used to understand the dynamics of EV-miRNA loading and identify the machinery involved in miRNA export. In this protocol, miRNAs predetermined to be enriched in EVs and depleted from donor cells are conjugated to a fluorophore and transfected into the donor cells. The fluorescently tagged miRNAs are then verified for loading into EVs and depletion from cells using qRT-PCR. As both a transfection control and a tool for gating the transfected population of cells, a fluorescently labeled cellular RNA (cell-retained and EV-depleted) is included. Cells transfected with both the EV-miRNA and cell-retained-miRNA are evaluated for fluorescent signals over the course of 72 h. The fluorescence signal intensity specific for the EV-miRNAs diminishes rapidly compared to the cell-retained miRNA. Using this straightforward protocol, one could now assess the dynamics of miRNA loading and identify various factors responsible for loading miRNAs into EVs.

作者聚焦：探索癌症进展中 MicroRNA 加载到细胞外囊泡中的机制 

Here, we describe a straightforward protocol that enables in vitro assessment of the abundance of fluorescently labeled microRNAs to study the dynamics of microRNA packaging and export into extracellular vesicles (EVs).

使用流式细胞术追踪miRNA释放到细胞外囊泡中

Extracellular vesicles (EVs) are important mediators of cellular communication that are secreted by a variety of different cells. These EVs shuttle ...

对于外小泡的使用流式细胞仪分析技术

摘要

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