对于外小泡的使用流式细胞仪分析技术
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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。 ?…
Representative Results
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…
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| LSR II benchtop flow cytometer | BD Biosciences | 3-laser (20 mW Coherent Sapphire 488 nm blue, 25 mW Coherent Vioflame 405 nm violet, and 17 mW JDS Uniphase HeNe 633nm red) | |
| FACS Diva software | BD Biosciences | PC version 6.0 | |
| FlowJo software | Treestar US | Mac version 9.6.1 or PC version 7.6.5 | |
| Sphero Rainbow fluorescent particles | BD Biosciences | 556298 | used to adjust all channel voltages to maintain fluorescence intensity consistency |
| Ultra Rainbow fluorescent particles | Spherotech | URFP-10-5 | used in addition to Megamix-Plus SSC beads to ensure EV gating consistency from batch to batch |
| Megmix-Plus SSC beads | Biocytex | 7803 | used to adjust FSC and SSC voltages to maintain consistency 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 |
| AbC Anti-Mouse Bead Kit | Life Technologies | A-10344 | used for compensation controls & negative AbC beads used for beads-based method |
| Nonidet P-40 Alternative (NP-40) (CAS 9016-45-9) | Santa Cruz | sc-281108 | 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. |
| BD TruCOUNT Tubes | BD Biosciences | 340334 | used whenver absolute EV concentrations are needed |
| Ultrafree-MC, GV 0.22 µm Centrifugal Filter Units | Millipore | UFC30GVNB | used to post-stain wash Evs and/or fractionate EVs based on size |
| Vacutainer glass whole blood tubes ACD-A | BD Biosciences | 364606 | |
| Facs tubes 12×75 polystrene | BD Biosciences | 352058 | |
| 50mL Reservoirs individually wrapped | Phenix | RR-50-1s | |
| Green-Pak pipet tips – 10µL | Rainin | GP-L10S | |
| Green-Pak pipet tips -200µL | Rainin | GP-L250S | |
| Green -Pak pipet tips – 1000µL | Rainin | GP-L1000S | |
| Stable Stack L300 tips presterilized | Rainin | SS-L300S | |
| Pipet-Lite XLS 8 Channel LTS Adjustable Spacer | Rainin | LA8-300XLS | |
| 96 well tissue culture plates | E&K Scientific | EK-20180 | |
| RPMI 1640 Media (without Hepes) | UCSF Cell Culture Facility | CCFAE001 | media used for bead-based detection method |
| Dulbeccos PBS D-PBS, CaMg-free, 0.2µm filtered | UCSF Cell Culture Facility | CCFAL003 | |
| Ultracentrifuge Tube, Thinwall, Ultra-Clear | BECKMAN COULTER INC | 344058 | |
| PANEL I | |||
| CD3 PerCP-Cy5.5 | Biolegend | 344808 | 2 µl |
| CD14 APC-Cy7 | Biolegend | 301820 | 2 µl |
| CD16 V450 | BD Biosciences | 560474 | 2 µl |
| CD28 FITC | biolegend | 302906 | 2 µl |
| CD152 APC | BD Biosciences | 555855 | 2 µl |
| CD19 A700 | Biolegend | 302226 | 2 µl |
| PANEL II | |||
| CD41a PerCP-Cy5.5 | BD Biosciences | 340930 | 2 µl |
| CD62L APC | Biolegend | 304810 | 2 µl |
| CD108 PE | BD Biosciences | 552830 | 2 µl |
| CD235a FITC | biolegend | 349104 | 2 µl |
| PANEL III | |||
| CD11b PE-Cy7 | Biolegend | 301322 | 2 µl |
| CD62p APC | Biolegend | 304910 | 2 µl |
| CD66b PE | Biolegend | 305106 | 2 µl |
| CD15 FITC | exalpha | X1496M | 5 µl |
| CD9 PE | Biolegend | 555372 | |
| CD63 APC | Biolegend | 353008 | |
| APC-Cy7 Ms IgG2a, κ | Biolegend | 400230 |
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Citer Cet Article
Inglis, H., Norris, P., Danesh, A. Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry. J. Vis. Exp. (97), e52484, doi:10.3791/52484 (2015).