We thank the following people: Jane Chu, Cameron Tran, James Lillard, Mafuz Khan, Masebonang Albert, Ken Rogers, and Syed A. Ali. Kateena Addae-Konadu was supported by UNCF/Merck Graduate Research Fellowship, American Medical Association Foundation, CRECD Grant 8R25MD007589-10, and NIH NIGMS MBRS Grant R25 GM058268. This work was supported by NIMHD grants 8G12MD007602, and 8U54MD007588, NIAID grant 1R21AI095150-01A1, Georgia Research Alliance grant GRA.VAC08.W, and Emory CFAR grant P30 A1050409.

外来体的分离和纯化过程的总体框图如图1中提供 。全血从健康志愿者的捐助者和未接受抗逆转录病毒theraoy参加埃默里大学的希望诊所和格雷迪健康系统的传染病计划HIV阳性者获得在佐治亚州亚特兰大。该研究获得埃默里大学医学院和莫尔豪斯学院的机构审查委员会。所有参与研究的人签署书面知情同意。 从血浆1.准备外来体的<ol><li> 人体血液采集与处理 <ol><li>收集10毫升外周血通过静脉穿刺在EDTA血液收集管（含有18毫克的钾EDTA）中，并轻轻颠倒5次混合。 </li><li>离心血液采集管在1000×g离心20分钟，在RT以沉淀血细胞。使用无菌吸管德的血浆部分（4-5毫升）转移到25毫升锥形管中。稀释血浆用10ml 1×PBS中。在对生物危险废物的适当标记的容器中丢弃血细胞（红细胞，白细胞，也被称为PBMC）。 注意:在未感染的供体的血液样品的情况下，PBMC可保留用于进一步使用（见步骤IV.2，下文）。 </li><li>储存血浆样品在4℃下短期（2-3天），或在-80℃下长期储存。 注:将冰冻的血浆样本，以4 摄氏度之前进一步处理。 </li></ol></li><li> 从血浆中的外来体分数的制备 <ol><li>离心机等离子体以10,000×g离心30分钟，在4℃以除去细胞碎片。使用无菌血清吸管给清零血浆上清液转移到一个干净25毫升超速离心管。丢弃在生物危害容器中的沉淀物。 </li><li>离心清等离子10万XG 2小时，在4℃，以除去大囊泡。小心地取出10万XG上清液吹打，并丢弃在生物危险废物。重悬在1ml的1X PBS的100000×g离心沉淀在一个干净的管中并在室温下孵育30分钟，轻轻地涡旋以移去和单独的颗粒。 </li><li>加入25毫升的PBS洗暂停100000 XG外来体/病毒颗粒。轻轻颠倒试管5次混合，然后再次离心以100,000×g离心2小时，在4℃。丢弃在生物危险废物容器中的PBS液冲洗。 </li><li>重悬100000×g离心沉淀在1ml 1×PBS中并在室温下孵育30分钟轻轻涡旋撞出和溶解沉淀。 注意:如果这是不可能直接进行到碘克沙醇梯度步骤，存储所述粒料，含有外来体和病毒颗粒，在4℃下1-2天，直到碘克沙醇梯度步骤。 </li></ol></li></ol> 2.净化外来体的<ol><li>生成6％-18％veloc碘克沙醇的使用双室梯度前装置，两者均梯度。 <ol><li>制备碘克沙醇试剂的6％和18％的溶液，供给作为在水中的60％的溶液中，通过稀释于PBS中。吸取5.5中毫升18％溶液进入搅拌室的，和吸管5.5中毫升6％溶液进入储存室。开启搅拌器，打开活塞，并且允许每个梯度流入14 ml的超速离心管。 注:要么立即使用或存放准备梯度O / N在4℃下才能使用。 </li><li>仔细层1毫升外来体/病毒溶液到每11个ml的梯度的顶端。离心机梯度在250000×g离心2小时，在4℃下，用SW40Ti吊桶式转子。 </li><li>标签12（12）1.5 ml微量离心管。从渐变的顶部除去1.0毫升并转移到管＃1中。转移顺序的其余1毫升馏分管2-12。 注:因此，最上面的级分将是＃1，底部馏分，＃12。圣矿石在4℃的梯度级分。纯化的外来体，其应在馏分1-3在梯度的顶部，贮存于4℃时是稳定的3-4周。 </li></ol></li></ol> 3.外来体的表征<ol><li> 乙酰胆碱酯酶活性测定 <ol><li>准备100毫米基板原料混合乙酰胆碱碘28.9毫克1毫升1X PBS中。商店基板股票在-20°C至1个月。 </li><li>准备10毫米彩色指标股混合苯甲酸39.6mg和碳酸氢钠15毫克于10毫升的1X PBS中。存储颜色指示剂股票在4℃下长达两个星期。 </li><li> 2:在100的比例制备测定试剂通过混合1×PBS，基板，以及彩色指示器5（例如，将10毫升的1X PBS + 200微升底物+500μl的颜色指示）。 </li><li>传送从各1.5 ml的梯度级分管（标记为1-12，从程序III.3）50微升到96孔microtite的孔- [R盘。准备两个孔的每个梯度样品。 </li><li>首先使2000μU/ ml的乙酰胆碱酯酶的股票在PBS准备一套标准。使11（11），2倍这个股票的连续稀释，并添加50微升每种稀释液至微量滴定板的单个孔，使得标准＃1 = 2000μU/毫升，＃2 = 1000μU/毫升，＃3 = 500μU/ ml的，等等，直到＃12 = 0.98μU/毫升。因此，12的AchE标准将占用一个96孔测定板的单个行。 </li><li>加入200μl测定试剂混合物的每个孔中，孵育20分钟（在黑暗中）在RT下，以使有色反应产物的发展。测量在450nm使用荧光酶标仪在波长AChE活性。 注意:在起动外来体纯化后的剩余材料的百分比是由普通等离子体胆碱酯酶法检测。 </li></ol></li><li> 免疫印迹分析 <ol><li>在梯度分数1-12分离蛋白（从的程序，URE 2.1.3）通过SDS-PAGE在4-20％的Tris-HCl预制凝胶在100V×1小时。在凝胶转移蛋白根据生产商的说明使用电印迹转移细胞硝酸纤维素膜上。允许转移到印迹12-16小时（O / N）为350V。 </li><li>从印迹装置取出膜和洗涤在Tris-缓冲盐水（TBS）中5分钟。用5％脱脂牛奶在TTBS（TBS含0.1％吐温20）1小时通过振荡在RT方框。 </li><li>孵育在5％脱脂牛奶特异于外来体（CD9，CD45，CD63）或HIV-1衣壳蛋白（p24的）第一抗体的膜，在4℃振荡12-16小时（O / N）。对于原发性免疫印迹稀释抗体的范围是从1:2000 -1:5000。 </li><li>用1洗印迹在TTBS 20分钟，然后孵育:2000稀释的辣根过氧化物酶（HRP）缀合的抗IgG抗体（二次抗体），在5％的脱脂牛奶1小时，在室温。洗净的印迹3次TTBS，每洗10分钟。 </li><lI&gt;孵育印迹用鲁米诺底物试剂根据制造商的指示，以产生化学发光信号从HRP标记蛋白检测使用具有CCD照相机的电子成像系统的化学发光信号。 </li><li>将图像保存为可以在Adobe Photoshop中查看TIFF文件。执行使用ImageJ软件（国立卫生研究院，马里兰州贝塞斯达）乐队的密度分析。 </li></ol></li><li> 细胞因子分析 <ol><li>在此之前的细胞因子测定，破坏免疫复合物，通常是存在于人血浆中的酸解离并裂解外来体通过去污剂处理。分析这两个启动血浆样品纯化外来体的准备。 <ol><li>至100μl人血浆添加100微升0.33当量盐酸，在37℃进行1小时。加入100微升0.33 1N氢氧化钠中和酸处理的血浆。添加的Triton X-100，以两者中和的血浆和纯化外来体的样品，给音响的1％的最终浓度，以引起外来体的裂解。 </li></ol></li><li>对于该测定法（荧光基于珠子的程序），使用磁珠预先涂覆有抗体由生产。使用抗人细胞因子抗体包被的珠的定制设计的面板; 在表1中列出。涡流从所述细胞因子测定试剂盒的抗体-包被的磁珠，30秒，并添加50微升珠96孔平板的每个孔在测定中使用。 </li><li>制备血浆标准缓冲液（均包含在细胞因​​子测定试剂盒）的稀释系列标准中的细胞因子测定试剂盒使用说明书中所述，以产生8点的标准曲线。 </li><li>添加150微升1X洗涤缓冲液（由试剂盒）到每个孔，并且洗涤用磁珠垫圈珠，如手册中所述。添加25微升血浆测定缓冲液（从盒）至各孔中。添加25微升标准品和样品，以在该测定中使用的所有孔中。加入25微升血浆样品缓冲液阴性对照孔中。密封，并在室温摇动板以700rpm进行60分钟和孵育O / N在4℃下。 </li><li>添加150微升1X洗涤缓冲液到每个孔，并且洗涤该板（如在步骤3.3.4，上文）。添加25μl的抗体检测到每个孔中，密封并在700rpm摇动板在室温30分钟。重复洗涤步骤。 </li><li>加入50微升链霉溶液（SAPE，从盒）到每个孔中，密封并在700rpm摇动板在室温30分钟。重复洗涤板在3.3.4。 </li><li>添加120微升读数缓冲液（从盒）到每个孔中，并摇动在实验室桌面摇床在700rpm进行5分钟，在室温以允许荧光信号来开发。读板放在读板器按制造商的说明。 </li><li>为了解释分离过程期间丢失外来体的量，可使用下列公式:[（原件读取/血浆体积）×66.6 =调节细胞因子读数。 </li></ol></li></ol> 4.检测方法的免疫调节潜力<ol><li> 培养基与外来体贫胎牛血清制备 <ol><li>离心机500毫升胎牛血清（FBS）中的10万xg离心20小时，在4℃以沉淀污染外来体和微泡。小心地取出并保存外来体耗尽FB​​S上清。丢弃在生物危险废物的颗粒。 </li><li>结合外来体贫FBS的上清液用500ml罗斯韦尔园区纪念研究所1640（RPMI 1640）培养基的20％。过滤通过0.45μm膜过滤器的介质。 </li><li>添加链霉素（100U /毫升），青霉素（100U /毫升），L-谷氨酰胺（2 mM计），HEPES缓冲的盐溶液（10μM），和IL-2（20U / ml）的经滤波的介质。 </li></ol></li><li> 细胞培养和外来体曝光 <ol><li>暴露外来体从健康诊所的志愿者得到的供体外周血单核细胞（PBMC）。在暂停PBMC制备培养基和计数使用细胞/颗粒计数器（根据制造商的标准方法）的细胞。 注:将PBMC上述等离子体制备过程中得到的（步骤1.2）。 </li><li>使用制备的介质从过程4.1.3，共培养3.0×10 6（PBMC）用1微克/毫升汇集外来体三（3）的HIV-1血清阳性或从三（3）的HIV-1血清反应阴性个体的总体积为1ml在12孔培养板（盖）的孔中。在37℃下孵育培养物48小时。 </li><li>准备与刀豆蛋白A处理未处理的外周血单个核细胞培养物和培养（ConA的; 5微克/毫升）以上（4.2.2），如作为阴性和阳性对照，分别。孵育所有PBMC培养48小时，在37℃。 </li><li>之前立即收集细胞，准备以下荧光标记的单克隆抗体的稀释度:的Alexa Fluor 700标记的抗CD3（1:400稀释），allophycocyANIN（APC）/花青7（Cy7的）标记的抗CD4（1:400），多甲藻素叶绿素蛋白复合物标记的抗CD4（1:400），V450标记的抗CD8（1:400），生物素标记的抗CD45RA（1:1000），藻红蛋白（PE）/ Cy7的标记的抗CD62L（1:1000），PE /花青5（Cy5的）标记的抗CD38（1:200），PE-德克萨斯红 - 标记的抗生蛋白链菌素（1:2000），和PE / Cy5标记小鼠IgG1K同种型对照（1:200）。 </li><li>在48小时的潜伏期后，收获PBMC。洗涤的PBMC在PBS中在4℃下温育除去外来体和细胞染色1小时与个别荧光染料标记的抗体。通过流式细胞仪分析染色的PBMC量化趋化因子的表达（如21）。 </li></ol></li></ol>

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<li>Lenassi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV+Nef+is+Secreted+in+Exosomes+and+Triggers+Apoptosis+in+Bystander+CD4+T+Cells%5BTitle%5D)+AND+%22Traffic%22%5BJournal%5D)">HIV Nef is Secreted in Exosomes and Triggers Apoptosis in Bystander CD4 T Cells.</a> Traffic. 11, 110-122 (2009).</li>
<li>Raymond, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV+Type+1+Nef+Is+Released+from+Infected+Cells+in+CD45%2B+Microvesicles+and+Is+Present+in+the+Plasma+of+HIV-Infected+Individuals%5BTitle%5D)+AND+%22AIDS+Res+Hum+Retrovir%22%5BJournal%5D)">HIV Type 1 Nef Is Released from Infected Cells in CD45+ Microvesicles and Is Present in the Plasma of HIV-Infected Individuals.</a> AIDS Res Hum Retrovir. 27, 167-178 (2011).</li>
<li>James, C. O., Huang, M. B., Khan, M., Garcia-Barrio, M., Powell, M. D., Bond, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+Nef+Protein+Targets+CD4%2B+T+Cells+for+Apoptosis+by+Interacting+with+CXCR4+Surface+Receptors%5BTitle%5D)+AND+%22Journal+of+Virology%22%5BJournal%5D)">Extracellular Nef Protein Targets CD4+ T Cells for Apoptosis by Interacting with CXCR4 Surface Receptors.</a> Journal of Virology. 78, 3099-3109 (2004).</li>
<li>Huang, M. B., Jin, L. L., James, C. O., Khan, M., Powell, M. D., Bond, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+Nef-CXCR4+Interactions+Important+for+Apoptosis+Induction%5BTitle%5D)+AND+%22Journal+of+Virology%22%5BJournal%5D)">Characterization of Nef-CXCR4 Interactions Important for Apoptosis Induction.</a> Journal of Virology. 78, 11084-11096 (2004).</li>
<li>Konadu, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Association+of+cytokines+with+exosomes+in+the+plasma+of+HIV-1-seropositive+individuals%5BTitle%5D)+AND+%22Journal+of+Infectious+Diseases%22%5BJournal%5D)">Association of cytokines with exosomes in the plasma of HIV-1-seropositive individuals.</a> Journal of Infectious Diseases. , (2014).</li>
<li>Cantin, R., Diou, J., Belanger, D., Tremblay, A. M., Gilbert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Discrimination+between+exosomes+and+HIV-1%3A+purification+of+both+vesicles+from+cell-free+supernatants%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Discrimination between exosomes and HIV-1: purification of both vesicles from cell-free supernatants.</a> J Immunol Methods. 338, 21-30 (2008).</li>
<li>Witwer, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standardization+of+sample+collection%2C+isolation+and+analysis+methods+in+extracellular+vesicle+research%5BTitle%5D)+AND+%22J+Extracellular+Vesicles%22%5BJournal%5D)">Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.</a> J Extracellular Vesicles. 2, 20360(2013).</li>
<li>Lässer, C., Eldh, M., Lötvall, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+Characterization+of+RNA-Containing+Exosomes%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Isolation and Characterization of RNA-Containing Exosomes.</a> J. Vis. Exp. (59), e3037(2012).</li>
<li>McDonald, M. K., Capasso, K. E., Ajit, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+and+microRNA+Profiling+of+Exosomes+Derived+from+Blood+and+Culture+Media%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media.</a> J. Vis. Exp. (76), e50294(2013).</li>
<li>Kanchi Ravi, R., Khosroheidari, M., DiStefano, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Modified+Precipitation+Method+to+Isolate+Urinary+Exosomes%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">A Modified Precipitation Method to Isolate Urinary Exosomes.</a> J. Vis. Exp. (95), e51158(2015).</li>
<li>Lötvall, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Minimal+experimental+requirements+for+definition+of+extracellular+vesicles+and+their+functions%3A+a+position+statement+from+the+International+Society+for+Extracellular+Vesicles%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles.</a> J Extracell Vesicles. 3, 26913(2014).</li>
</ol>

The authors declare that they have no competing financial interests.

慢性免疫激活（CIA）和CD4 + T细胞耗竭是HIV-1感染的两个重要特征。他们已经被确立为预测的发病机制，与美国中央情报局是最好的预测。然而，驾驶慢性全身免疫激活和CD4 + T细胞衰退的作用机制还没有完全阐明。我们和其他的实验室已经开发出坚定的证据表明，外来体从HIV-1感染细胞分泌起到两个标志的作用。 在两种组合物和细胞外囊泡的功能继续关心导致了各种方法外来?...

在HIV-1流行继续在世界各地都有一个显著的影响。截至2013年，全球约有3500万人感染艾滋病毒，而这些210万新感染的个体1。预防战略和增加获得抗逆转录病毒疗法已在减少整体收购艾滋病毒有帮助。然而，个别人群仍面临上升收购HIV 1。因此，有必要继续努力来解决这一流行病。 其中一个HIV疾病进展的最强预测的是慢性免疫激活（CIA）2-10。持续高检测水平的细胞因子和T淋巴细胞的表面上表达升高的标记限定，CIA已被归因于:i）连续的树突状细胞产生I型IFN 11的类型; （二）直接免疫激活HIV蛋白达，NEF和gp120的12驱动; （三）易位相关的肠道免疫细胞6细菌蛋白质。然而，确切的机制（S）潜在慢性，在HIV感染的全身免疫激活仍有待完全阐明。 我们的研究小组和其他人证明外来体的艾滋病发病15-18的作用。我们小组已确定的Nef蛋白从感染的细胞中分泌的外来体15，和外来体的Nef（exNef）存在的HIV感染的个体中的纳克水平18在血浆中。我们已经表明，旁观者的CD4 + T细胞暴露于exNef导致活化诱导的细胞死亡取决于CXCR4途径19，20，另外，单核细胞/巨噬细胞难治exNef诱导的细胞凋亡，但表现出改变的细胞功能和细胞因子的表达。最近，我们集团已经显示出外来体从HIV感染者的血浆中分离出含有多种促炎细胞因子。福rther，诱导CD38表达对天真和中央记忆CD4 +和CD8 + T细胞的HIV感染患者暴露于血浆来源的外来天真的外周血单核细胞。这可能有助于全身性炎症，并通过旁观者细胞活化21病毒的繁殖，并表明外来体发挥在HIV发病机理中显著作用。 在调查外来体在HIV发病机理中的作用，一个挑战是发展技术以来自HIV颗粒，同时保持外体含量有效地分离外泌体，以及它们的功能免疫调节能力。几种方法已经被描述用于从细胞培养物和生物流体22,23分离外泌体。因为它们的小尺寸和低的密度（外来体漂浮在1.15的密度- 1.19克/毫升），差分超速离心和/或超滤是最常用的技术为外来体隔离23。但是，艾滋病毒感染细胞培养上清和病人的血浆同时包含外来体和HIV-1病毒颗粒。外来体和HIV-1的颗粒在尺寸和密度非常相似。另外，以独特的外体标记，如CD63，CD45，CD81和表达的优势，外来体一直在使用免疫亲和捕获方法23隔离。这个过程可以从外来体分离病毒。然而，这种技术的缺点是紧密附接抗体与纯化的外来体，这可能干扰在培养外来体的免疫调节的潜能的评估。 为了解决这些局限性，我们开发了用于使用碘克沙醇的速度梯度来自HIV颗粒在人血浆中由Cantin改性和同事22外来体的纯化过程。外来体被发现在低浓度偏析碘克沙醇梯度/上层级分，而病毒颗粒分离在高密度/较低的分数。病毒颗粒通过P24 ELISA检测和使用外来体标记的乙酰胆碱酯酶，CD9，CD63和CD45被确定外来体。收集上层低密度部分包含这是不含有HIV-1 P24污染外来体。艾滋病毒颗粒在人体血浆中外来体的高效纯化和分离方法可以从人血浆以及他们的免疫调节潜力的调查和外来体在HIV-1发病机制中的诊断和预后价值来源的外来内容的准确检查。

<table><tbody><tr><td>BD Vacutainer EDTA tubes (10ml)&amp;nbsp;</td><td>Becton Dickinson</td><td>368589</td><td>pink top tubes</td></tr><tr><td>Lymphoprep Ficoll reagent</td><td>Cosmo Bio</td><td>AXS-1114545</td><td></td></tr><tr><td>Optiprep iodixanol reagent</td><td>Sigma</td><td>D1556</td><td></td></tr><tr><td>14ml ultracentrifuge tubes</td><td>Beckman Coulter</td><td>344060</td><td>ultraclear tubes</td></tr><tr><td>Gradient Former Model 485</td><td>BIO-RAD</td><td>165-4120</td><td></td></tr><tr><td>Acetylthiocholine iodide</td><td>Sigma</td><td>1480</td><td></td></tr><tr><td>Benzoic Acid</td><td>Sigma</td><td>D8130</td><td></td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Acetyl Cholinesterase</td><td>Sigma</td><td>C3389</td><td></td></tr><tr><td>96-well clear microtiter plate</td><td>Medical Supply Partners</td><td>TR5003</td><td></td></tr><tr><td>SpectraMax 190 microplate reader</td><td>Molecular Devices</td><td>190</td><td>Fluorescent plate reader</td></tr><tr><td>Criterion Gel Electrophoresis Cell</td><td>BIO-RAD</td><td>165-6001</td><td></td></tr><tr><td>Transblot Gel&amp;nbsp;</td><td>BIO-RAD</td><td>170-3910</td><td></td></tr><tr><td>Transfer Cell</td><td></td><td></td><td></td></tr><tr><td>Tris-HCl Criterion precast gels</td><td>BIO-RAD</td><td>567-1093</td><td></td></tr><tr><td>Anti-CD45 antibody&amp;nbsp;</td><td>Abcam&amp;nbsp;</td><td>Ab10558</td><td></td></tr><tr><td>CD63 Antibody (H-193)</td><td>Santa Cruz Biotech, Inc.</td><td>SC-15363</td><td></td></tr><tr><td>CD9 Antibody (H-110)</td><td>Santa Cruz Biotech, Inc.</td><td>SC-9148</td><td></td></tr><tr><td>Rabbit pAb p24 HIV-1&amp;nbsp;</td><td>ImmunoDX, LLC</td><td>1303</td><td></td></tr><tr><td>Nitrocellulose membrane&amp;nbsp;</td><td>BIO-RAD</td><td>G1472430</td><td></td></tr><tr><td>Tris Buffered Saline</td><td>BIO-RAD</td><td>170-6435</td><td></td></tr><tr><td>HRP-conjugated IgG (H+L) secondary antibody</td><td>Thermo Scientific</td><td>31460</td><td>Goat-Anti-Rabbit</td></tr><tr><td>HRP-conjugated IgG (H+L) secondary antibody</td><td>Thermo Scientific</td><td>31430</td><td>Goat-Anti-Mouse</td></tr><tr><td>Western Blotting Luminol Reagent</td><td>Santa Cruz Biotech, Inc.</td><td>SC-2048</td><td></td></tr><tr><td>GE LAS-4010 Imager</td><td>GE Healthcare</td><td>LAS-4010</td><td></td></tr><tr><td>Human Procarta Cytokine Immunoassay Kit</td><td>Affymetrix&amp;nbsp;</td><td>N/A</td><td>Custom immunoassay panel</td></tr><tr><td>Bio-Plex 200 Immunobead Reader</td><td>BIO-RAD</td><td>171-000201</td><td></td></tr><tr><td>Coulter Z2 Particle Counter</td><td>Beckman Coulter</td><td>383552</td><td>Cell counter</td></tr><tr><td>Alexa Fluor 700-labeled anti-CD3</td><td>BD Bioscience (UCHT1)</td><td>300424</td><td></td></tr><tr><td>APC/Cy7-labeled anti-CD4</td><td>Biolegend (OKT4)</td><td>317418</td><td></td></tr><tr><td>PerCP-labeled anti-CD4</td><td>BD Bioscience (RPA-T8)</td><td>550631</td><td></td></tr><tr><td>V450-labeled anti-CD8</td><td>BD Bioscience (RPA-T8)</td><td>560347</td><td></td></tr><tr><td>Biotin-labeled anti-CD45RA</td><td>BD Bioscience (HI100)</td><td>555487</td><td></td></tr><tr><td>PE/Cy7-labeled anti-CD62L</td><td>Biolegend (DREG-56)</td><td>304822</td><td></td></tr><tr><td>PE/Cy5-labeled anti-CD38</td><td>Biolegend (HIT2)</td><td>303508</td><td></td></tr><tr><td>APC/Cy7-labeled anti-HLADR</td><td>Biolegend (L243)</td><td>307618</td><td></td></tr><tr><td>PE-Texas Red-labeled anti-streptavidin</td><td>BD Bioscience</td><td>551487</td><td></td></tr><tr><td>PE/Cy5-labeled mouse IgG1K</td><td>Biolegend (MOPC-21)</td><td>400116</td><td></td></tr><tr><td>APC/Cy7-labeled mouse IgG2aK</td><td>Biolegend (MOPC-173)</td><td>400229</td><td></td></tr></tbody></table>

isolation of exosomes from the plasma of hiv-1 positive individuals

外来体是小囊泡从从它们的多种细胞类型的释放都组成型和刺激后30纳米至100纳米不等的大小。他们发现，在一些生物体液的和已知携带多​​种蛋白质，脂质和核酸分子。原本以为要多一点水库细胞碎片，外来体调节生物过程和疾病中的作用也越来越赞赏。 几种方法已经被描述用于分离从细胞培养基和生物流体的外来体。由于它们的小尺寸和低的密度，差分超速离心和/或超滤是最常用的技术为外来体隔离。然而，HIV-1感染的个体的血浆中含有两个外来体和HIV病毒颗粒，其在尺寸和密度相似。因此，从人血浆中的HIV病毒颗粒的外来体的有效的分离已一个挑战。 为了解决此限制，我们开发了从Cantin 等改性的过程。 人，2008为来自HIV颗粒在人血浆中的外来体的纯化。碘克沙醇的速度梯度被用于外来体来自HIV-1颗粒的HIV-1阳性个体的血浆中分离。病毒颗粒确定P24 ELISA。外来体被确定的外来体标记物的乙酰胆碱酯酶的基础（乙酰胆碱酯酶）和CD9，CD63和CD45抗原上。我们的渐变过程产生游离病毒颗粒的外来体的准备。从人血浆外来体的高效净化使我们能够检查血浆衍生的外来体的含量，并调查它们的免疫调节潜力和其他生物学功能。

外泌体有效地从HIV-1阳性的人血浆纯化。分离的外来体，确定乙酰胆碱酯酶的活性，分隔在较低密度组分1-3在碘克沙醇梯度的顶部，而病毒颗粒，确定HIV-1抗原的p24 ，隔离在高密度级分（10-12，底部附近）。外来体的存在下通过的外来体标记物的AChE，CD9，CD45，CD63和，并用电子显微镜（图2）的免疫印迹鉴定进一步证实。 <p class="jove_content" fo:keep...

Watch this Scientific Journal Video about Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals at JoVE.com

从 HIV-1 阳性个体血浆中分离外泌体 |免疫学和感染|视频

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

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

从HIV-1阳性者的血浆分离外来体的

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

isolation-of-exosomes-from-the-plasma-of-hiv-1-positive-individuals

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SCIENTISTS IN ACTION

17.3K 次观看.  Morehouse School of Medicine. Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

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

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

高密度脂蛋白的分离的非编码小RNA定量

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

科研

生物化学

Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral and cellular mucosal immune system1,2. 
 Microbiota is shielded from the immune system via a strong mucosal barrier. Infections and antibiotics are known to alter both the normal 
	gastrointestinal tract barrier and the composition of resident bacteria, which may result in possible immune abnormalities3.
HIV causes a breach in the gastrointestinal barrier with progressive failure of mucosal immunity and leakage into the systemic circulation of bacterial bioproducts, such as lipopolysaccharide and 
	bacterial DNA fragments, which contribute to systemic immune activation4-7. Microbial translocation is implicated in HIV/AIDS immunopathogenesis and response to therapy 4,8.
We aimed to characterise the composition of bacteria translocating in peripheral blood of HIV-infected patients. To pursue our aim we set up a PCR reaction for the panbacteric 16S ribosomial gene 
	followed by a sequencing analysis.
Briefly, whole blood from both HIV-infected and healthy subjects is used. Given that healthy individuals present normal intestinal homeostasis no translocation of microflora is expected in 
	these patients. Following whole blood collection by venipuncture and plasma separation, DNA is extracted from plasma and used to perform a broad range PCR reaction for the panbacteric 
	16S ribosomial gene9. Following PCR product purification, cloning and sequencing analyses are performed.

Our experiment will show how to perform a sequencing analysis of bacterial species translocating in peripheral blood of HIV positive patients.

测序外周血中细菌菌群：我们的经验与艾滋病毒感染者

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral ...

医学

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

蛋白质消化、超滤和尺寸排除色谱法优选人血浆和血清外来体的分离

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.

从HIV-1感染细胞的外来体基于SILAC蛋白质组学表征

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

免疫与感染

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

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

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

分离和剖析人力胆汁含外来体的MicroRNA-

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

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA含外来体的分离和鉴定

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

生物学

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

高产细胞外囊泡制剂的纯化远离病毒

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

MicroRNAs play an important regulatory role and are emerging as novel therapeutic targets for various human diseases. It has been shown that miRNAs are carried in high density lipoproteins. We have developed a simplified method to rapidly isolate purified HDL suitable for miRNA analysis from human plasma.

从高纯高密度脂蛋白的miRNA的高通量快速分离和简化方法

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for ...

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing,&#160;microarrays and can be&#160;utilized in functional in vitro/in vivo studies.

<meta charset="utf8"></head><body>使用过滤和超速离心分离和表征人脂肪细胞来源的细胞外囊泡

We describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

使用过滤和超速离心分离和表征人脂肪细胞来源的细胞外囊泡

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can ...

从HIV-1阳性者的血浆分离外来体的

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使用过滤和超速离心分离和表征人脂肪细胞来源的细胞外囊泡