This work of AA, SZ, WF, J-C.G, and LM was supported by the NICHD Intramural Program. The work of MV was supported by the Russian Federation Government grant #14.B25.31.0016.

1.耦合磁性纳米粒子（的MNP） <ol><li> 使用市售偶合方法和试剂以耦合的MNP与抗体（Abs）以下（通常为1毫克）。与羧酸反应性基团的氧化铁纳米颗粒被使用。 <ol><li>如果抗体是在一个体积大于0.5毫升，集中使用100K集中器。转速为2000 XG为3-5分钟。 </li></ol></li><li>在该过程结束时，加入10微升猝灭缓冲液后，的MNP转移到12mm的×75毫米管和加入3 1ml洗涤/存储缓冲器。放置管在磁性分离器的中心孔，并在4℃下离开过夜。 </li><li>验证的MNP都收集到管的一侧，然后小心地吸取了所有液体。加入3毫升的新鲜洗涤/存储缓冲器，并放回磁铁。 </li><li>几个小时之后，检查是否的MNP被收集在管的一侧，然后吸管出液体。用2个1ml洗涤/存储缓冲器来悬浮的MNP，并在4℃下保存。 </li><li>验证绝对通过用相关的荧光Fab抗体片段标记它们（如果使用小鼠单克隆抗体用于捕获如在第2描述例如山羊抗-小鼠），耦合到的MNP和流式细胞仪上运行。 </li></ol> 2.标记抗体再加到的MNP <ol><li>结合60微升（3.9×10 12颗粒）的MNP（每条件）和5微升（200微克/毫升的商业溶液）标记的Fab片段，特异于捕获Ab的在1.5ml微量离心管中的同种型。 </li><li>在室温下孵育（RT）下连续搅拌30分钟。 </li><li>在1500 xg离心预润湿100K聚光用300μl磷酸盐缓冲盐水（PBS）和自旋在微量5分钟。 </li><li>净化100K上标记列配合。自旋从步骤2.2的混合物在微量在1500×g离心5分钟，用200μlPBS洗涤，并在恢复初始音量。 </li></ol> 3.使用标记AB-MNP配合物的捕捉利益颗粒（病毒或电动汽车） <ol><li> 孵育预标记的MNP 60微升（3.9×10 12颗粒）与HIV（60微升）或EV制剂（100微升）。 <ol><li>准备使用各种方法EV准备。在这里，对净化蔗糖梯度电动车，采用外切体沉淀剂从贫血小板血浆收集它们，或直接从血浆分离 8。 注:最佳比率需要为每个实验进行确定，并且依赖于在制备病毒/ EV的浓度。 MNP抗体比例应为10〜6过剩相比，病毒/ EV分数的浓度。 </li></ol></li><li>孵育在37℃下温和混合40分钟的病毒，或在4℃下1小时对电动汽车。 </li><li>添加2.5％的鼠IgG /人IgG阻断Fc结合，轻轻涡旋，在室温孵育3-5分钟。 </li><li>将每个检测的ABS制造商推荐或滴定浓度，并在室温下孵育额外的20分钟。 </li></ol> 4.分离从自由抗体的MNP捕获的病毒粒子（或电动车）使用磁列<ol><li>通过将它放置在一个分离器磁铁准备使用磁性分离柱。 </li><li>预湿用500μl洗涤缓冲液（2mM的乙二胺四乙酸（EDTA），0.5％牛血清白蛋白（BSA）的PBS）的列。允许洗涤缓冲液通过柱流动。 </li><li>在MNP病毒/ MNP-EV复合物添加到列，并允许所有的液体流过。添加500μl的洗涤缓冲液对柱子，使整个卷通过。 </li><li>用洗涤缓冲液2次以上重复洗涤。 </li><li>宽度仅为12 mm×75毫米管留用MNP病毒/ MNP-EV复合物的收集取下磁铁和地点列。让柱立在管从磁铁3分钟。添加的PBS 200微升，并让珠粒流下通过重力，添加另外200微升的PBS，并用洗脱后200微升4％多聚甲醛固定。 注意:多聚甲醛是一种可疑的致癌物，应在通风橱中处理和手套应戴。 </li><li>为了量化单病毒颗粒/电动汽车上使用高通量取样器（HTS）或之前，为了增加收购充分混合流式细胞计数珠体积设置。 </li></ol> 5.的MNP捕获病毒的分析/用流式细胞仪电动车<ol><li>预热流式细胞仪30分钟。 </li><li>运行质量控制珠。 </li><li>在任的MNP或标示病毒/电动汽车的荧光设定的阈值。使用0.22微米的过滤PBS阈降低背景"噪音"。通过在水平调整PMT电压设定的阈值，过滤PBS没有给出，或者很少，事件。 </li><li>低运行样品。 （使用串联设置背后为鞘液额外0.04微米在线过滤器标准的0.2微米的过滤套，进一步消除假事件）。 </li></ol> 6.捕获效率评价<ol><li>在3.1,3.2描述捕捉荧光标记的电动汽车或HIV与MNP-ABS。 （电动车可被标记或用膜的染料，或通过他们的货物 15;病毒可通过基因编码的染料16标记之一或用膜染料12，17）。 </li><li>如（4.1-4.2）的介绍准备磁选柱。 </li><li>在MNP病毒/ MNP-EV复合物添加到列，并允许所有的液体通过流（流过分数）。收集通过分数在管中的流动。 </li><li>用如（4.4-4.5）所述保留在柱上馏分进行。 </li><li>从6.3与随后的分离（4.1-4.5）通过分数的流动重复采集过程。 </li><li>分析从第一CA保留部分通过运行它们的流式细胞仪和pture第二捕获设置触发电动汽车或HIV的荧光标签上。为了评估效率，比较在这两个级分的事件的数量。 </li></ol> 7.适应议定书特定任务的注意:虽然上述的协议可应用于HIV和电动汽车的分析，对于DENV的分析下列修改应引入: <ol><li>孵育用1μM的DiI病毒体在黑暗中在RT 30分钟。温育后在24万xg离心纯化染色病毒通过在密度梯度介质离心（10％，20％，25％和35％），在4℃下没有制动1.5小时。收集20％和25％的密度梯度介质层14之间部分。 </li><li>孵育DiI标记DENV 1×10 7（60微升），在2.5％的小鼠IgG的存在下，在室温20分钟检测Ab的制造商推荐或滴定浓度。 </li><li> Incub吃了预标记的60微升（3.9×10 12颗粒）的MNP捕获抗体复合物混合物在37℃下温和混合40分钟。 </li><li>与未结合的绝对分离所得复合物在以上所描述和流式细胞仪上流动分析磁列。 </li></ol>

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The authors declare no conflict of interest.

常规的流式细胞仪保持用于分析根据其物理和化学特性在个人层面18细胞中的主要工具。流的基本原则术以来没有它的发展改变了:穿过激光束和散射光的单元构成触发通过细胞结合的荧光抗体的荧光光谱的记录的事件。低于300纳米的更小的颗粒不能被细胞仪的侧向散射，因为它们根据在大多数流式细胞仪不收集光18更大的角度散射更多的光被检测到。这里描述的技术，可以识别和多种抗原的众多个体病毒或与电动车引发的荧光分析用传统的流式细胞仪。 该协议中的关键步骤是抗体的MNP的耦合。我们的实验perforMED使用具有羧酸作为反应性基团为15nm的氧化铁纳米颗粒如前所述 9。根据制造商，每次15纳米MNP最多可以20抗体的结合;我们绑定约为每股15纳米MNP 10抗体。这一估计是基于抗体前，偶联后的数量和由纳米粒子特性和尺寸测量仪器珠/毫升的数量。原则上，耦合可以引起颗粒的聚集，并且不希望的。要检查聚合与我们的协议发生的事情，我们与纳米粒子特性和尺寸的仪器验证了这一点。此仪器测量的粒子的流体动力学直径，其中包括该涂层和抗体的层，而不是只在15纳米的内核。我们发现，大多数的偶联珠位于一个单峰（在64.7±4.2纳米平均峰值低于73±7.3纳米的所有事件的90％）。 AB-MNP合作的混合在正确的比例病毒颗粒或电动汽车mplexes很重要，这样的MNP是几个数量比电动车/高病毒粒子的浓度。这是至关重要的，以确保单个电动汽车/病毒体进行了分析。通过在低流式细胞仪运行避免了事件的巧合。确保触发荧光的获取和使用体积控制来测量捕获的实体的浓度。 但是，的MNP可以通过交联或由两个或更多病毒体的相同的纳米颗粒的结合聚集电动汽车或病毒颗粒。要进行检查，如在图4中描述为聚集的测试已被执行。甚至单独捕获病毒颗粒或电动车可以同时流式细胞仪输入流的激光束。这两种不同的抗原创造假阳性。为了避免这种情况，如所描述的流式细胞仪应设置和制剂应进行稀释。一种可以检查重合是否正在发生通过使用相同的如前面的点描述的策略。此外，缺乏一致的可以通过分析制备的连续稀释液，并证明事件的数量，而一个荧光染色抗原的平均荧光强度保持在所有稀释液（ 图5）恒定线性稀释减小进行验证。另一个问题是，太少病毒/电动车可以被捕获。这可能是由于病毒/携带通过它们由特定的MNP捕获抗原电动汽车的真正稀缺。备选地，捕获的效率是低的，由于各种原因（ 例如 ，抗体的MNP，从的MNP的至病毒体/电动汽车此协议制定的比率偏差等的低效耦合）。避免了后者的可能性，捕获的效率应具体评价。典型地，该效率应为约90％，如在图6中 。 不同的抗体可由于干扰位阻彼此或与耦合到的MNP的抗体。要检查这种情况没有发生反向捕捉协议必须进行测试（如第7简单地说，病毒粒子或电动车与随后的分离从自由浮动的绝对描述应与荧光检测绝对先染色，然后通过捕捉AB-MNP复合磁列。 流virometry具有相对于病毒粒子或电动汽车的抗原性组合物的分析的现有方法显著优点。常规生化方法对特定蛋白质的筹备普遍出现了汇报，但所提供的病毒粒子或电动车的异质性的信息。这包括大型商用颗粒病毒体或电动车的免疫捕获。这样的颗粒典型地在直径为几微米，他们每个人的可捕获的病毒颗粒或电动汽车的数百或数千。因此，任何后续的分析平均病毒粒子/电动汽车的性能一个粒子捕获。与此相反，流动virometry使用的10-15纳米的MNP的直径，并与所述协议的至少90％的事件表示单个病毒粒子或单一的EV。 主要的限制是，在制备病毒体或电动汽车的整个人口可能不被分析，但只有那些被捕获。因此，通过该病毒粒子或电动汽车被捕获的抗原的选择变得至关重要。此外，与流式细胞术不同抗原的数量（针对不同抗原的荧光抗体的数目）由病毒粒子或电动汽车的小面的限制。最后，不同的抗体可在空间上相互再次干扰由于小（相对于细胞）表面。 目前的协议可适于任何病毒或任何来源的电动车的分析，只要通过它们可以被捕获的特异性抗体可用。对个别病毒颗粒许可证抗原分析的研究人员，以解决病毒人群的不同组分的发病机制。此外，在等离子体个别电动车辆的识别使得可以跟踪电动车到特定细胞的起源和关于这些细胞的正常或病理状况和在它们所在的器官的报告。

在多细胞生物体的细胞有许多单个功能可以是唯一的一个特定的细胞或至少属于细胞唯一的组。即使克隆培养的细胞表现出个人的特点其形态多样证明。另外，一个单元的个性体现在它分泌的产物。在病毒感染的情况下，病毒颗粒可以携带从产生它们的细胞中的蛋白质。例如，对于HIV许多宿主细胞蛋白质被嵌入病毒包膜和由不同的细胞产生的病毒可能携带这些特性的蛋白质1,2或"小区签字"。 即使没有感染，细胞释放到周围介质的小囊泡外（EVS）。这些囊泡和它们在许多情况下，结构的生物合成表现得像是病毒，特别是逆转录病毒。近年来它已成为明确该电动汽车，它最初被认为是"血小板尘"3，构成细胞间通讯的一个重要的生理系统。连同间通讯，即细胞-细胞接触的相互作用和释放可溶性分子的其它两个系统，电动汽车坐标正常生理和在各种病症4,5包括病毒感染6,7改变。 虽然这两个释放病毒颗粒和细胞外囊泡是高度多样的，它们是由在其中它们各自的特性丧失散装分析主要特征。类似于流式细胞术的方法，其中表型根据其不同的表面抗原的细胞，需要表征个体的病毒和病毒样颗粒。不幸的是，电动车或小病毒颗粒无法使用标准流量分析CYTOmetry方法，因为它们太小，以产生在其上最血细胞计数依靠用于触发光散射信号。规避此限制，荧光触发可以适用。然而，如果电动汽车或病毒颗粒与荧光抗体染色也难以从由于其类似的荧光和可比尺寸的自由抗体及其聚集体区分开来。 最近，我们开发了基于纳米技术的高通量方法，允许使用常规的流式细胞仪8,9个人的小颗粒抗原的特性。我们使用的MNP到感兴趣免疫捕获的颗粒，如已被其它基团为不同的应用10，11，12，13中所述;然而，我们的新技术允许单个特定的捕获其次FIC目标由这些捕获的粒子的表型通过流式细胞仪用荧光触发，而不是光散射，而不自由浮动抗体的干扰。该方法具有广泛的应用，并且可以用于表征在体内被细胞产生的任何viral-和非病毒小颗粒的抗原性组合物，并在体外提供针对表面抗原的特异性荧光抗体的可用性。我们已经应用这个技术来研究生物学的几个问题。 特别是，我们评估了对个体的HIV-1颗粒9的宿主细胞标记物的分布，我们研究了基于它们的表面蛋白14的分析个别登革热病毒（DENV）的成熟和调查释放到健康志愿者和患者的血流中的电动汽车急性冠状动脉综合征（ACS）。虽然基于类似的原理，在这种新技术的应用所需的那下面描述特定协议的开发。

<table border="0" cellpadding="0" cellspacing="0" width="868">
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		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Materials</td>
			<td style="width:164px;"></td>
			<td style="width:119px;"></td>
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			<td style="width:164px;">Ocean Nanotech</td>
			<td style="width:119px;">ICK-15-005</td>
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			<td style="width:164px;">Millipore</td>
			<td style="width:119px;">UFC810024</td>
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			<td style="width:164px;">Falcon</td>
			<td style="width:119px;">352002</td>
			<td></td>
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			<td height="21" style="height:21px;width:423px;">DEPC treated water</td>
			<td>Quality Biological&#160;</td>
			<td style="width:119px;">351-068-131</td>
			<td></td>
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			<td height="21" style="height:21px;">SuperMAG-01 magnetic separator&#160;</td>
			<td style="width:164px;">Ocean Nanotech</td>
			<td style="width:119px;">SuperMAG-01</td>
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			<td height="18" style="height:18px;">Zenon R-Phycoerythrin Mouse IgG1&#160;Labeling Kit</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">Z25055</td>
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			<td height="21" style="height:21px;">Nanosep Centrifugal Devices with Omega Membrane 100k</td>
			<td style="width:164px;">Pall Corp</td>
			<td>OD100C34</td>
			<td></td>
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			<td height="21" style="height:21px;width:423px;">EDTA 0.5M</td>
			<td style="width:164px;">Corning Cellgro</td>
			<td style="width:119px;">46-034-CI</td>
			<td></td>
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			<td height="21" style="height:21px;">Bovine Serum Albumin solution</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td>A9576-50ML</td>
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			<td height="21" style="height:21px;">PBS</td>
			<td style="width:164px;">Gibco</td>
			<td style="width:119px;">10010-23</td>
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			<td height="21" style="height:21px;">Paraformaldehyde, EM Grade, Purified</td>
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			<td style="width:119px;">15710</td>
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			<td height="21" style="height:21px;">123count eBeads&#160;</td>
			<td style="width:164px;">eBioscience</td>
			<td>01-1234-42</td>
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			<td height="21" style="height:21px;width:423px;">CytoCount beads</td>
			<td style="width:164px;">Dako</td>
			<td>S2366</td>
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			<td height="21" style="height:21px;width:423px;">AccuCheck count beads</td>
			<td style="width:164px;">Invitrogen</td>
			<td style="width:119px;">PCB100</td>
			<td></td>
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			<td height="21" style="height:21px;width:423px;">Cytometer Setup &#38; Tracking Beads Kit (CST)</td>
			<td>BD Bioscience</td>
			<td>642412</td>
			<td></td>
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			<td height="21" style="height:21px;width:423px;">Vybrant DiI Cell-Labeling Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>V22885</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
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			<td>Catalog Number</td>
			<td>Comments</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Equipment</td>
			<td style="width:164px;"></td>
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			<td height="21" style="height:21px;">&#181;MACS column&#160;</td>
			<td style="width:164px;">Miltenyi Biotec</td>
			<td>130-042-701</td>
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			<td height="21" style="height:21px;width:423px;">OctoMACS Separator magnet</td>
			<td style="width:164px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-042-108</td>
			<td></td>
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			<td height="21" style="height:21px;width:423px;">Nanodrop</td>
			<td style="width:164px;">ThermoFisher</td>
			<td style="width:119px;">none</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:423px;">NanoSight NS300</td>
			<td style="width:164px;">Malvern</td>
			<td style="width:119px;">none</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:423px;">BD LSRII flow cytometer</td>
			<td style="width:164px;">BD Bioscience</td>
			<td style="width:119px;">none</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:423px;">Software</td>
			<td style="width:164px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">Flowjo software</td>
			<td>Flowjo</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">BD FACSDiva software</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

由HIV-1病毒颗粒的细胞抗原捕获选择用"流virometry"现在是可能的可视化在个别病毒颗粒的细胞的抗原。作为一个例子，我们侧重于HIV-1，LFA-1和HLA-DR进行两个蜂窝抗原。此前这两种抗原散装1分析HIV-1的准备进行了鉴定。我们制备的MNP加上抗包膜病毒的蛋白质，VRC01的一种抗体，以及与这些的MNP的HIV-1病毒粒子（HIV-1 LAI.04或HIV-1 SF162在外周血单核细胞（PBMC）产生的）捕获。此外，我们染色这些病毒粒子与2G12，另一种抗包膜抗体。流virometry表明对于LFA-1的和HLA-DR的存在下的HIV-1病毒粒子的高异质性。平均16.7±2.0％（N = 6）和8.6±0.3％（N = 3）中进行的LFA-1和HLA-DR的HIV-1 LAI.04病毒体，分别。只有4.8±0.6％（N = 3）所有的病毒粒子呈阳性两种抗原。另外HIV-1毒株，HIV-1 SF162，这些抗原存在于20.0±4.4％（6例）和10.8±1.3％（N = 6）病毒粒子，分别，而这两种抗原是由6.5±进行0.4％（ 图1）。 的细胞蛋白的分布依赖于复制的病毒的细胞。感染艾滋病毒的病毒颗粒生产从这些被感染外周血单个核细胞产生的抗原性不同的Jurkat细胞。由Jurkat细胞产生的HIV-1 LAI 0.4的病毒粒子进行1.6±1.4％（N = 3）分别和1.6±0.7％（N = 4）的LFA-1和HLA-DR。在Jurkat细胞产生的HIV-1 SF162病毒粒子，这些参数分别为7.2±2.5％（N = 3）和5.6±2.7％（N = 4）。因此，抗原性化妆（至少对HLA-DR和LFA-1）不同的HIV-1 LAI.04由两个不同的细胞产生的类型（P &lt;0.02）。 流virometry应用于登革病毒的评价 DENV成熟与prM蛋白对病毒体的表达相关联。我们采用流virometry评估什么BHK-1产生的登革病毒的分数，并在LoVo细胞构成成熟的病毒。病毒粒子与油脂染料染色的DiI。透露个人捕获的病毒粒子分析的prM在48.2±5.3％（N = 8）DENVs的。相反，84.5±3.4％（N = 4）DENV的中携带的prM LoVo细胞产生的。病毒体的其余部分，分别51.8±5.3％（N = 8）和15.5±3.4％（N = 4）分别的prM阴性（ 图2）。因此，流virometry可以用来区分未成熟（或部分成熟）DENV病毒体完全成熟个体DENV。 外囊泡释放到健康的血液志愿者和急性冠状动脉综合征（ACS） 为了研究在ACS患者和健康对照不同的EV子集，我们通过荧光的MNP耦合用抗CD31，CD41a，或CD63的抗体捕获从贫血小板血浆（PPP）的电动汽车。通过CD31-的MNP捕获电动汽车进行染色CD41a和CD63，由CD41a-的MNP捕获电动汽车进行染色CD31和CD63，以及CD63-的MNP捕获电动汽车进行染色CD31和CD41a（ 图3）。 电动汽车用CD31-的MNP和阳性ACS患者的检测抗体的一个或两个所捕获的量为3,359 [2328; 5,472]电动车/μL在1,272 [714比较; 2157]电动车/μl，以健康志愿者（P = 0.001）。电动汽车与健康志愿者比较ACS患者由CD63捕获的总金额为3541 [1318; 5173]电动车/μL与806 [488; 2,112]电动车/μL（P = 0.007）。有4,752 [3238; 7,173]电动车/μL在ACS患者通过CD41a-的MNP捕获等离子，而在健康志愿者血浆这个数字是显著降低，2,623 [1927; 4,188]电动车/μL（P = 0.015）。一般情况下，我们的研究结果表明，虽然电动汽车量的ACS患者多为增加，增加的幅度是电动车的不同亚群不同。 <img alt="图1" src="/files/ftp_upload/55020/55020fig1.jpg" /> 图1:HIV-1病毒体在不同细胞类型中复制细胞抗原的选择性结合。艾滋病毒（HIV-1 LAI.04或HIV-1 SF162）通过的PBMC或Jurkat细胞释放病毒粒子与VRC01-的MNP被捕获，并与第二抗-gp120抗体2G12与针对HLA-DR和LFA-1的特异性抗体染色。染色的准备经受了HLA-DR（白条）和LFA-1（黑巴流量分析RS）。装置的三到六个实验9±SEM表示。 <a href="http://ecsource.jove.com/files/ftp_upload/55020/55020fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55020/55020fig2.jpg" /> 图2:登革病毒颗粒的成熟状态。在生产DENV BHK-21细胞（A，B），或在LoVo细胞（C，D）分别标以脂质染料的DiI和，在密度梯度介质超速离心后，染色prM蛋白与2H2抗体（A，C）或与同型对照的IgG2a（B，D）。标记的病毒颗粒然后用3H5-1-的MNP捕获并进行流动分析14。 <a href="http://ecsource.jove.com/files/ftp_upload/55020/55020fig2large.jpg" target="_blank">请点击此处查看这个大版本</a>数字。 <img alt="图3" src="/files/ftp_upload/55020/55020fig3.jpg" /> 图3:ACS患者和健康对照电动车的分析。等离子电动汽车用CD31-的MNP，CD63-的MNP或CD41a-的MNP抓获，并针对CD41a和CD63，CD31反对和CD41a和分别对CD31和CD63抗体染色。捕获电动车，与检测绝对的至少一种染色，进行可视化，并在流分析列举。数据以中位数和四分位距（IQR）点图。绿色符号:健康志愿者（对照组），棕色符号:ACS患者。 <a href="http://ecsource.jove.com/files/ftp_upload/55020/55020fig3large.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/55020/55020fig4.jpg" /> 图4:Evaluati上表示单个病毒粒子流事件的分数。 （A），HIV-1。病毒颗粒的悬浮液分成两部分并染色要么了DiD（左图）或DIO（ 中图 ）。在混合后，该包含了两个差动染病毒颗粒的悬浮液，用VRC01-的MNP捕获并经受流动virometry（ 右图 ）。聚集体（双色事件）构成的事件总数的小于10％。 （B - D）登革病毒。的DiI染病毒粒子的悬浮液连续稀释为1两倍:2至1:256和流式细胞（B）的与超导获得。 的DiI-DENVs用2H2抗体（C）或的DiI-DENVs标有3H5-1-的MNP（D）被抓获。制剂C和D然后连续稀释为1两倍:2至1:256，并与高温超导14获得的。病毒颗粒似乎并没有在所有情况下，因为那里聚集是稀释因子和事件的数量之间的线性相关性。 <a href="http://ecsource.jove.com/files/ftp_upload/55020/55020fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55020/55020fig5.jpg" /> 图5: 单颗粒通过流式细胞仪检测。的DiI染色DENV悬浮液连续稀释为1两倍:2至1:2048和一个流使用高温超导仪获得的。 （A）的检测作为稀释因子的函数病毒数量。 （B）的中值荧光强度（MFI）的DiI标记的DENV 14。 <a href="http://ecsource.jove.com/files/ftp_upload/55020/55020fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <i毫克ALT ="图6"SRC ="/文件/ ftp_upload / 55020 / 55020fig6.jpg"/&gt; 图6:病毒粒子和与耦合到特异性抗体的MNP捕获外囊泡的分数的评价。标记的HIV-1病毒颗粒或电动车用的MNP连接到特定的抗体，并进行流virometry被抓获。上磁列分离后，流过（不保留）级分进行分析病毒粒子或感兴趣电动车的存在下错过了在第一次运行这一点。 （A）标记的HIV-1病毒颗粒BAL用2G12-的MNP（ 左图）抓获。流过级分回收并进行流动virometry（ 右图）。在第一次循环的感兴趣的病毒粒子的约95％被抓获。 （B）的标记电动车用抗CD81的MNP上捕获，分离磁列（左面板）。流过级分重新捕获并分析（ 右图）。在第一感兴趣的囊泡的周期约99％的被抓获8。 <a href="http://ecsource.jove.com/files/ftp_upload/55020/55020fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles at JoVE.com

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

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 ...

flow-virometry-to-analyze-antigenic-spectra-virions-extracellular

Research

JoVE Journal

Immunology and Infection

7.8K 次观看.  Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health. 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.

视频: Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

嗜中性粒细胞胞外的陷阱：如何生成和可视化

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

科研

免疫与感染

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

使用"单个病毒基因组的单病毒颗粒的分离和基因组分析&#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

一种优化的协议分析和糖酵解线粒体呼吸的淋巴细胞

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

支气管肺泡灌洗小鼠肺分析炎症细胞浸润

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

成年日本血吸虫细胞外囊泡的分离与鉴定

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

Antigenic Liposomes for Generation of Disease-specific Antibodies

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a na&#239;ve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

抗原脂质体用于生成特定于疾病的抗体

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

利用超滤离心技术分离小鼠支气管肺泡灌洗液中的细胞外囊

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...