Name: flow virometry to analyze antigenic spectra of virions and extracellular vesicles
Uploaded: 2017-01-25T15:00:00.000+00:00
Duration: 6 min 28 s
Description: Watch this Scientific Journal Video about Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles at JoVE.com

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>

<ol>
	<li>Tremblay, M. J., Fortin, J. F., Cantin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acquisition+of+host-encoded+proteins+by+nascent+HIV-1.">The acquisition of host-encoded proteins by nascent HIV-1.</a> Immunol Today. 19 (8), 346-351 (1998).</li><li>Santos, S., Obukhov, Y., Nekhai, S., Bukrinsky, M., Iordanskiy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-producing+cells+determine+the+host+protein+profiles+of+HIV-1+virion+cores.">Virus-producing cells determine the host protein profiles of HIV-1 virion cores.</a> Retrovirology. 9, 65 (2012).</li><li>Wolf, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+and+significance+of+platelet+products+in+human+plasma.">The nature and significance of platelet products in human plasma.</a> Br J Haematol. 13 (3), 269-288 (1967).</li><li>Piccin, A., Murphy, W. G., Smith, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microparticles:+pathophysiology+and+clinical+implications.">Circulating microparticles: pathophysiology and clinical implications.</a> Blood Rev. 21 (3), 157-171 (2007).</li><li>Bernal-Mizrachi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+levels+of+circulating+endothelial+microparticles+in+patients+with+acute+coronary+syndromes.">High levels of circulating endothelial microparticles in patients with acute coronary syndromes.</a> Am Heart J. 145 (6), 962-970 (2003).</li><li>Rozmyslowicz, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-+and+megakaryocyte-derived+microparticles+transfer+CXCR4+receptor+to+CXCR4-null+cells+and+make+them+susceptible+to+infection+by+X4-HIV.">Platelet- and megakaryocyte-derived microparticles transfer CXCR4 receptor to CXCR4-null cells and make them susceptible to infection by X4-HIV.</a> Aids. 17 (1), 33-42 (2003).</li><li>Bhattarai, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GB+virus+C+particles+inhibit+T+cell+activation+via+envelope+E2+protein-mediated+inhibition+of+TCR+signaling.">GB virus C particles inhibit T cell activation via envelope E2 protein-mediated inhibition of TCR signaling.</a> J Immunol. 190 (12), 6351-6359 (2013).</li><li>Arakelyan, A., Ivanova, O., Vasilieva, E., Grivel, J. C., Margolis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenic+composition+of+single+nano-sized+extracellular+blood+vesicles.">Antigenic composition of single nano-sized extracellular blood vesicles.</a> Nanomedicine. 11 (3), 489-498 (2015).</li><li>Arakelyan, A., Fitzgerald, W., Margolis, L., Grivel, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle-based+flow+virometry+for+the+analysis+of+individual+virions.">Nanoparticle-based flow virometry for the analysis of individual virions.</a> J Clin Invest. 123 (9), 3716-3727 (2013).</li><li>Yang, H., Liang, W., He, N., Deng, Y., Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemiluminescent+labels+released+from+long+spacer+arm-functionalized+magnetic+particles:+a+novel+strategy+for+ultrasensitive+and+highly+selective+detection+of+pathogen+infections.">Chemiluminescent labels released from long spacer arm-functionalized magnetic particles: a novel strategy for ultrasensitive and highly selective detection of pathogen infections.</a> ACS Appl Mater Interfaces. 7 (1), 774-781 (2015).</li><li>Tang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+and+rapid+detection+of+Pseudomonas+aeruginosa+based+on+magnetic+enrichment+and+magnetic+separation.">Highly sensitive and rapid detection of Pseudomonas aeruginosa based on magnetic enrichment and magnetic separation.</a> Theranostics. 3 (2), 85-92 (2013).</li><li>Borlido, L., Azevedo, A. M., Roque, A. C., Aires-Barros, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+separations+in+biotechnology.">Magnetic separations in biotechnology.</a> Biotechnol Adv. 31 (8), 1374-1385 (2013).</li><li>Xi, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+HBsAg-Specific+DNA+Aptamers+Based+on+Carboxylated+Magnetic+Nanoparticles+and+Their+Application+in+the+Rapid+and+Simple+Detection+of+Hepatitis+B+Virus+Infection.">Selection of HBsAg-Specific DNA Aptamers Based on Carboxylated Magnetic Nanoparticles and Their Application in the Rapid and Simple Detection of Hepatitis B Virus Infection.</a> ACS Appl Mater Interfaces. 7 (21), 11215-11223 (2015).</li><li>Zicari, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+maturation+of+individual+Dengue+virions+with+flow+virometry.">Evaluation of the maturation of individual Dengue virions with flow virometry.</a> Virology. 488, 20-27 (2016).</li><li>Lai, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+and+tracking+of+tumour+extracellular+vesicle+delivery+and+RNA+translation+using+multiplexed+reporters.">Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters.</a> Nat Commun. 6, 7029 (2015).</li><li>McDonald, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+intracellular+behavior+of+HIV+in+living+cells.">Visualization of the intracellular behavior of HIV in living cells.</a> J Cell Biol. 159 (3), 441-452 (2002).</li><li>Morcock, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elimination+of+retroviral+infectivity+by+N-ethylmaleimide+with+preservation+of+functional+envelope+glycoproteins.">Elimination of retroviral infectivity by N-ethylmaleimide with preservation of functional envelope glycoproteins.</a> J Virol. 79 (3), 1533-1542 (2005).</li><li>Shapiro, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical Flow Cytometry. , 101-223 (2005).</li></ol>

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><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Carboxyl Magnetic Iron Oxide Nanoparticles Conjugation Kits</td><td>Ocean Nanotech</td><td>ICK-15-005</td><td></td></tr><tr><td>Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-100 membrane</td><td>Millipore</td><td>UFC810024</td><td></td></tr><tr><td>5 ml Round Bottom PP Test Tube, without Cap</td><td>Falcon</td><td>352002</td><td></td></tr><tr><td>DEPC treated water</td><td>Quality Biological&amp;nbsp;</td><td>351-068-131</td><td></td></tr><tr><td>SuperMAG-01 magnetic separator&amp;nbsp;</td><td>Ocean Nanotech</td><td>SuperMAG-01</td><td></td></tr><tr><td>Zenon R-Phycoerythrin Mouse IgG1&amp;nbsp;Labeling Kit</td><td>Thermo Fisher Scientific</td><td>Z25055</td><td></td></tr><tr><td>Nanosep Centrifugal Devices with Omega Membrane 100k</td><td>Pall Corp</td><td>OD100C34</td><td></td></tr><tr><td>EDTA 0.5 M</td><td>Corning Cellgro</td><td>46-034-CI</td><td></td></tr><tr><td>Bovine Serum Albumin solution</td><td>Sigma-Aldrich</td><td>A9576-50ML</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010-23</td><td></td></tr><tr><td>Paraformaldehyde, EM Grade, Purified</td><td>EMS</td><td>15710</td><td></td></tr><tr><td>123 count eBeads&amp;nbsp;</td><td>eBioscience</td><td>01-1234-42</td><td></td></tr><tr><td>CytoCount beads</td><td>Dako</td><td>S2366</td><td></td></tr><tr><td>AccuCheck count beads</td><td>Invitrogen</td><td>PCB100</td><td></td></tr><tr><td>Cytometer Setup &amp;amp; Tracking Beads Kit (CST)</td><td>BD Bioscience</td><td>642412</td><td></td></tr><tr><td>Vybrant DiI Cell-Labeling Solution</td><td>Thermo Fisher Scientific</td><td>V22885</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;micro;MACS column&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-701</td><td></td></tr><tr><td>OctoMACS Separator magnet</td><td>Miltenyi Biotec</td><td>130-042-108</td><td></td></tr><tr><td>Nanodrop</td><td>ThermoFisher</td><td>none</td><td></td></tr><tr><td>NanoSight NS300</td><td>Malvern</td><td>none</td><td></td></tr><tr><td>BD LSRII flow cytometer</td><td>BD Bioscience</td><td>none</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Flowjo software</td><td>Flowjo</td><td></td><td></td></tr><tr><td>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

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 ...

科研

免疫与感染

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 ...

生物化学

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

病毒融合动力学测量方法在单粒子能级

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

生物学

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

替代体外测定病毒衣壳结构完整性的方法

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

<meta charset="utf8"></head><body>使用双报告基因和多重技术分析 SARS-CoV-2

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

<meta charset="utf8"></head><body>作者聚焦:通过新型免疫捕获和质谱技术推进抗病毒策略

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

通过多重双报告基因策略分析病毒颗粒上的表面蛋白表位

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

与VirusMapper超分辨率显微镜开源单粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Single-Particle Interferometric Reflectance Imaging Characterization of Individual Extracellular Vesicles and Population Dynamics

Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different biological molecules, including protein, lipid, DNA, and RNA, and are postulated to facilitate cell-to-cell communication in diverse tissues and organs. Recently, EVs have attracted significant attention as biomarkers for diagnostics and therapeutic agents for various diseases. Many methods have been developed for EV characterization. However, current methods for EV analysis all have different limitations. Thus, developing efficient and effective methods for EV isolation and characterization remains one of the crucial steps for this cutting-edge research field as it matures. Here, we provide a detailed protocol outlining a single-particle interferometric reflectance imaging sensor (SP-IRIS), as a method that is capable of detecting and characterizing EVs from unpurified biological sources and purified EVs by other methodologies. This advanced technique can be used for multi-level and comprehensive measurements for the analysis of EV size, EV count, EV phenotype, and biomarker colocalization.

This protocol presents single-particle interferometric reflectance imaging that is designed for the multi-level and comprehensive measurements of extracellular vesicles (EV) size, EV count, EV phenotype, and EV biomarker colocalization.

流量Virometry来分析病毒颗粒的抗原谱和外囊泡

摘要

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此视频中的章节

使用“单个病毒基因组的单病毒颗粒的分离和基因组分析'

基于荧光的人体血液单细胞外囊快速表征

病毒融合动力学测量方法在单粒子能级

替代体外测定病毒衣壳结构完整性的方法

单病毒原子力显微镜和超分辨率荧光成像的样品制备

通过多重双报告基因策略分析病毒颗粒上的表面蛋白表位

通过多重双报告基因策略分析病毒颗粒上的表面蛋白表位

通过多重双报告基因策略分析病毒颗粒上的表面蛋白表位

与VirusMapper超分辨率显微镜开源单粒子分析

Single-Particle Interferometric Reflectance Imaging Characterization of Individual Extracellular Vesicles and Population Dynamics