我们感谢马库斯·塔利，弥敦道罗伊，克里斯托弗·齐格勒，艾米莉布鲁斯，便雅悯王为通过赠款T32 AI055402（JK），R21 AI088059（JB），以及P20RR021905有益的探讨和美国国立卫生研究院支持这些研究（JB） 。

注意:这是非常关键的，所描述的协议，采用严格的预PCR技术（见28关于如何建立一个-PCR实验室前和如何使用好-PCR技术前进行实验的精彩概述）进行。至关重要的是，所有的试剂和仪器都是免费的含有定量PCR靶序列扩增的DNA，并有适当的控制到位，以检测这种污染。该协议的概述示于图1。 1.准备在48孔板媒体和种子细胞<ol><li>通过加入50ml制备含有10％胎牛血清，1％青霉素 - 链霉素500毫升完整的Dulbecco改良Eagle培养基中（DMEM）中，和1％的HEPES（N-2-羟乙基-N-2-乙烷磺酸）缓冲液的热灭活胎牛血清和5ml每一个100X青霉素 - 链霉素和100X HEPES缓冲溶液到500ml瓶中的DMEM组成。该试剂可以被存储在476℃持续几个月。 </li><li>种子2.5×10 4个 Vero E6细胞每孔在500μl的完全DMEM的最终体积的48孔组织培养板。孵育在37℃的板在含5％CO 2的10至18小时的湿润培养箱。 <ol><li>在工作​​日的最后种子板块。测试在任一复制或三孔每个病毒样本。 注意:虽然用于本测定基于48孔平台被描述，但应该有可能向下使用缩放测定在96-或384-孔板中。 </li></ol></li></ol> 2，开展病毒细胞附着注意:在这个协议中使用的病毒，JUNV应变偷拍＃1（C＃1）中，已成功地用作用于预防JUNV疾病29的减毒活疫苗。 JUNV C＃1，从通过在体内和体外连续传代的毒力JUNV应变XJ衍生和由12个氨基酸30亲XJ菌株不同。因为...ËJUNV C＃是1级生物安全（BSL）-2级病原体，本节介绍的工作，必须使用适当的BSL-2实践31进行。这包括使用个人防护装备（ 例如眼睛保护，实验室外套，双层手套），II级生物安全柜，并确保所有人员都收到了机构培训，并安全地处理这个有机体所需的批准。 <ol><li>稀释JUNV C＃1样品（多个）进行测试，以所希望的PFU就能或者在冰冷完全DMEM焦点形成单位并存储在冰上直到准备添加到每个孔中。 注意:给导电与病毒在完全DMEM稀释结合研究另一种选择，使用含有PBS的基本培养基具有降低的血清浓度（2％）和合适的缓冲液来建立血清是否可能会导致与病毒附着干扰。替代病毒类似附件协议已报道没有bicarbo使用RPMI 1640培养基内特含有0.2％牛血清白蛋白，10mM的（2-（吗啉代）乙磺酸）和10mM HEPES，pH 6.8的32。这种结合媒体可能优于防止当介质从培养箱中的CO 2 -environment除去可能发生的pH变化。 <ol><li>接种与PFU就能井从200至2,000，以限制非生产性附着事件。正如图2所示，该测定能够利用少至20 PFU（或多达2×10 6 PFU）检测附件。 </li><li>创建病毒的预混接种到细胞上。每种病毒的样本将在每孔50μl的体积接种到重复的孔中。因此，如果每孔病毒的2000 PFU就能增加是需要的，稀释4800 PFU就能为120微升冰的总体积冷完成DMEM中。 <ol><li>为了控制用于测定的特异性，使用一对匹配的中国仓鼠卵巢细胞系的，要么不表达TFR1（TRVb）或EXPRESS hTfR1（TRVb-1），为JUNV附着到这些细胞要么应不分别13,33减少或。另外，治疗用蛋白酶的细胞，如蛋白酶K，以防止沙粒病毒附件34或入境35。可溶性hTfR1或单克隆抗体ch128.1，这是具体 ​​的hTfR1，也可以考虑，因为它们是公知的方框JUNV的进入细胞13,36。此外，免费的甘露糖14或特定JUNV中和抗体37颗粒孵化预处理细胞可以抑制病毒进入。 </li><li>但是，请注意，这些试剂后者和方法，尽管拦截JUNV项，尚未得到确认阻止的附件，虽然这是一个可能的解释。此处所描述的协议可用于正式确定这些各种方法的影响病毒体附着。 </li></ol></li></ol></li><li>从37℃培养箱中取出盘子和放置或者在4℃冰箱或在冰上15分钟以抑制铺板的细胞来进行细胞内吞作用的能力。 </li><li>接着，吸出从每个完全DMEM井，并使用100μl的冰冷的PBS（磷酸盐缓冲盐水），PH值7.4的迅速洗每个孔两次。然后加入50微升预稀释的病毒样品（在步骤2.1中制备）向适当孔中。 </li><li>包裹板保鲜膜，并立即将要么回到冰上或4℃冰箱中1〜1.5小时，以允许进行病毒附件。保持板，洗涤缓冲液，和病毒的样品冷在4℃贯穿此步骤。 </li><li>在4℃温育完成后，吸出病毒接种物，并用200微升冰冷的PBS洗每个孔3次。 </li></ol> 3.病毒RNA提取<ol><li>通过使用商业试剂盒（ 例如 ，的RNeasy Mini试剂盒）雅从JUNV C＃纯化病毒RNA附着在单层1颗粒鼎制造商的协议。具体来说，按照上中的RNeasy Mini手册（ 第 4版，2006年4月）下面列出的修改页面23-28"使用旋转技术的协议动物细胞的总RNA净化"。 <ol><li>裂解如通过直接添加350微升裂解缓冲液RLT中向每个孔朝向步骤1B中的细胞。混合缓冲几次，以确保细胞溶解。注意，细胞容易在该缓冲溶解和完全裂解可通过倒置显微镜下观察孔进行验证。 </li><li>转移得到的细胞裂解物的试剂盒，柱（步骤3a）中，并按照协议的其余部分所描述的。在这一点上，该病毒已被裂解缓冲液中和这样的协议的其余步​​骤可在II级生物安全柜的外面来完成所提供的试剂盒管不沾染感染性病毒。 </li><li>包括来自制造商的方案，这是一个ADDIT可选步骤9旋转柱的有理离心来消除缓冲液RPE的任何可能残留。 </li><li>在最后的步骤中，制造商的方案（步骤10），洗脱使用30微升无核酸的DDH 2 O的旋转柱纯化的RNA的在-80°C储存RNA在这一点上，或直接在定量RT-PCR检测使用。为了防止病毒纯化RNA样品降解核酸酶，使用无核酸试剂和设备，并保持解冻RNA应放在冰上。 注意:缓冲器RLT和RW1含有胍盐，不应与漂白剂混合。缓冲RW1含有乙醇。 </li></ol></li></ol>病毒基因组的4定量RT-PCR检测<ol><li>转换JUNV C＃1 S片段的RNA成cDNA用于随后的qPCR，若使病毒RNA（在步骤3.1.4生成）至RT在50微升的总体积。 <ol><li>要设置RT反应，首先创建一个包含各单位反应以下试剂预混液:6.75微升核酸-free DDH 2 O的，5微升2微米的库存的RT引物的S的2098-到2075-（5&#39;-AAGGGTTTAAAAATGGTAGCAGAC-3&#39;），这是向S段基因组RNA的NP区域，5微升的互补10X PCR缓冲液II，11微升的25mM MgCl 2溶液，1.25微升（62.5单位）逆转录酶，RNA酶抑制剂的1微升（0.4单位），以及10微升500μM的股票的dNTP的。 注意:在沙粒病毒复制的过程中，产生了4病毒S片段RNA种类:全长基因组RNA（vRNA节），全长基因组RNA（vcRNA）（即vRNA节的反向互补）和两个亚基因组mRNA的编码NP和GP基因，分别为。这里列出的RT引物是特异于仅S片段vRNA节，但不是vcRNA，NP的mRNA，或GP的mRNA。 </li><li>轻轻地混合RT预混液，然后分装40微升成单个0.2毫升PCR管。添加10微升病毒RNA至每个管。包括:i）一个无模板对照管（ 如:。添加无核酸酶DDH 2 O的40微升主混合物）和ii）一个没有RT酶对照的10微升（ 例如加10微升已知阳性病毒RNA样品到主混合物的40微升未接受任何逆转录酶）来筛选对RT试剂与含有病毒的靶序列扩增的DNA的污染。包括来自未感染的细胞与完整的RT主混合物，以控制在细胞RNA的存在下测定特异性提取的RNA。 </li><li>此外，包括已知的阳性病毒RNA样品充分预混，以验证对RT试剂的质量。注意，RT反应的体积可以根据需要降低到25微升。 </li></ol></li><li>若使RT管，以下面的循环条件:25℃，10分钟，48℃30分钟，和95℃5分钟。注意，样品可以通过加入4℃的步骤中保存在-20℃，在​​这一点上，或在热循环仪放置过夜。 </li><li> Perform的qPCR在25微升的总体积。 <ol><li>做出的qPCR主混合物通过混合下述:每2.5微升（9μM股票）的正向PCR引物S1937 +可1958+（5&#39;-CATGGAGGTCAAACAACTTCCT-3&#39;）和反向PCR引物小号2001-到1982-（5所述的qPCR探针小号1960+的&#39;-GCCTCCAGACATGGTTGTGA-3&#39;），2.5微升（2μM股票）至1975+（5&#39;-6FAM-ATGTCATCGGATCCTT-MGBNFQ-3&#39;），和12.5微升2X通用qPCR主混合。注意，正向引物这里报告，这是具体 ​​的JUNV C＃1，由1的不同核苷酸从最初报告引物序列38，其靶向有毒JUNV应变Romero的。 </li><li>轻轻地混合溶液中，然后加入20微升到每个定量PCR好。添加5μl的每个RT反应，以适当的定量PCR孔中。进行定量PCR中一式三份进行测试各样品。请注意，如果需要的PCR反应体积可减小到低至12.5微升。 注意:与相关联的软件在定量PCR机将每个未知样品的值进行比较，以一系列编码JUNV C＃1 NP基因，这是定量PCR引物的靶质粒的标准稀释物产生JUNV C＃1 S片段的基因组RNA的绝对拷贝数和探头设置。的qPCR分析只能为落入标准曲线范围内的值提供定量。出于这个原因，包括质粒下列数量（一式三份的孔）:5，50，5×10 3，每5×10 5和5×10 7个拷贝良好。 </li></ol></li><li>加载的qPCR板进入热循环和运行以下反应条件:95℃，10分钟，15秒和60℃1分钟的95℃的40个循环。 <ol><li>收集和分析的数据，根据制造商的协议来确定每个样品中的病毒RNA的存在量。 </li></ol></li><li>要使用质粒含有焦油的qPCR新纯化后的样品建立标准曲线得到的序列，首先确定在该溶液中的质粒拷贝数。 <ol><li>计算出的质粒的分子量。如果整个质粒的序列是已知的，计算该使用以下公式:分子量=（[A * 312.2] + [G * 328.2] + [C * 288.2] + [T * 303.2] - 61.13）。请记住，包括对双链质粒的每条链中发现特定核苷酸的总数。 </li><li>如果该质粒的大小是已知的，但它的确切序列不是，计算出各双链DNA碱基对具有600兆瓦的假设下的兆瓦。 注:这里使用的将pCAGGS-JUNV C＃1 NP质粒具有的4.2×10 6兆瓦。 </li><li>一旦该质粒的MW已经确​​定，计算每微升多少副本存在于库存质粒溶液。 </li><li>先计算的质粒的总微克在该溶液中，然后将其转换为质粒的摩尔（ 例如 ，如果69微克质粒都包含在300微升的水中，然后6.9×10 -5μg质粒* 1摩尔/ 4.2×10 6微克质粒的=质粒1.6×10 -11摩尔），它可以被转换成拷贝数（ 例如质粒的1.6×10 -11摩尔* 6.022×10 23分子/摩尔= 9.8×10 12分子（或复印件））。 </li><li>通过该溶液的体积除以在质粒溶液质粒的总拷贝得到每微升份（ 例如 9.8×10 12拷贝/ 300微升= 3.28×10 10个拷贝）。 </li><li>适当稀释该溶液，使5微升体积可以装载到PCR板传递以建立标准曲线所需拷贝数的范围内。 </li></ol></li></ol>

<ol>
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作者宣称，他们没有竞争的经济利益。

我们描述的协议是简单的和健壮提供了以下关键的考虑因素观察。首先，严格预-PCR技术必须使用（参见第28一个很好的回顾）。至关重要的是，所有的试剂和仪器都是免费的含有定量PCR靶序列扩增的DNA，并有适当的控制到位，以检测这种污染。第二，对于协议，病毒，细胞和介质的病毒 - 细胞附着部分必须在4℃下保持，以防止表面结合的病毒粒子的内吞作用。第三，收集用于RNA提取的细胞的数量必须不超过RNA纯化滤波器的RNA结合的能力。第四，纯化的病毒RNA样品必须通过使用不含核酸酶的试剂和设备，并通过解冻时使它们保持在冰上免受核糖核酸酶介导的降解。第五，标准曲线的范围包括在qPCR分析必须足够大捕获来自未知样品观察到的所有的值。第六，技术重复应包括用于测定两者的病毒 - 细胞附着部以及单个RNA样品的定量PCR。可以包括第七，控制（ 如细胞表达hTfR1与否线）来控制用于 ​​测定的特异性。最后，所有人员必须具备相应的生物安全培训和机构的批准，以处理感染JUNV C＃1或其他致病性沙粒病毒。 协议的qPCR的部分要求一个的qPCR探针主要是为了确保在PCR反应的每个阶段被放大的产物的特异性。测定的成本可以然而减少，通过有利于另一个qPCR的化学不需要的探针消除这种qPCR的探针（ 如利用该非对称染料SYBR Green I的25）和/或通过减少的体积使用qPCR的反应混合物。如果探针定量PCR的独立应用程序蟑螂被使用，以验证该反应条件是不够严格保证的定量PCR的引物的特异性这将是很重要的。同样重要的是要知道，沙粒病毒的RNA，无论是从细胞或病毒颗粒中提取，可以非特异性引物，以形成对RT期间的cDNA在不存在病毒特异性RT引物12的。因此，复制使用vRNA的特异RT引物没有严格反映刚刚通过RT引物所针对的vRNA节种检测数字。如果基因组中特异性或反基因是必需的，我们已经报道通过使用生物素化的RT引物和链霉珠12绕过这个非特异性吸现象的装置。 此法的一个重要优势是它的极端敏感性。其他沙粒病毒细胞附着试验具有放射性标记的22，20生物素或荧光标记的21颗粒需要0.2的MOI，100或1000，各自LY。虽然使用的放射性标记颗粒的结果良好的灵敏度，它必须使用放射性的，这增加了与该特定测定法相关的后勤复杂性和安全性问题。这里描述的基于定量PCR法需要少至20 PFU（的MOI〜0.0004的48孔板）并能够可靠地检测附着在的MOI的一个大的动态范围（从0.0004 如的MOI至至少40）（ 图3 ）。该测定的强敏感性使得有可能使用低的MOI用于通过标准的感染性病毒的生产结合事件的更精确的测量。高灵敏度也显著减少所需用于测定病毒的量，并且在这样做时，限制了在测定中所用可能影响病毒粒子的质量的额外处理步骤（病毒体如基于聚乙二醇的沉淀，标记的病毒颗粒与荧光团或放射性同位素和/或virio的蔗糖带NS）。此外，qPCR分析本身很简单执行以及高度准确，重复性好。 所述细胞附着测定可以进行修改，以测量病毒进入包括病毒颗粒的内吞作用以及病毒融合的最后一步，也就是释放病毒基因组进入细胞质的附加阶段。为了测量颗粒的内吞作用，连接协议的相同的步骤，将通过步骤2.5，这是颗粒附着到细胞在4℃下执行。然后细胞将被加热到允许病毒颗粒被内吞，随后用蛋白酶剥离如所述22,24的未内吞任何外部结合的病毒粒子。部分3和该协议的4然后将遵循以提取病毒RNA和定量病毒基因组RNA的拷贝作为内吞作用的量度。为了测量基因组脱壳以下病毒和体膜融合，细胞将与被预先绑定病毒在4℃下，加热（以允许内吞作用和融合），除去外部颗粒，然后收集用于分析。在一种方法中，细胞可以在等渗缓冲液中裂解，以保持细胞内囊泡，然后分成2部分:一个是将与核糖核酸酶的混合物（未涂覆病毒RNA是易于降解）被处理，而另一个是不会39 。备选地，细胞可以被分离成细胞质或内体部分40。在这两种情况下，定量RT-PCR将用于定量病毒基因组在细胞质中作为病毒基因组脱壳的量度。最后，在定量RT-PCR测定可以容易地修改，以研究病毒附着，内吞作用，或融合所提供的，RT-PCR试 ​​剂针对感兴趣的新病毒等病毒（例如参见24-27）。

沙粒病毒是那些主要保持在啮齿类动物中自然1-3笼罩，单链RNA病毒家族。虽然这些病毒通常建立在啮齿动物水库1,2无症状，持续感染，可导致人类3严重疾病。重要的致病品种包括新世界胡宁病毒（JUNV）4和旧世界拉沙病毒5，分别是阿根廷出血热在南美和拉沙热的病原体在非洲。淋巴细胞脉络丛脑膜炎病毒（LCMV），家庭6的原型病毒，具有全球分销并负责多种疾病状态，包括在免疫活性个体6无菌性脑膜炎，在发育中的胎儿7严重的先天缺陷，或在免疫抑制高致死以下固体器官移植8,9个人。缺乏美国食品和药物管理局（FDA）批准的疫苗或打击这些病毒有效的抗病毒药物凸显迫切需要制定新的策略来治疗针对这些新兴/再出现的病原体。 的沙粒病毒基因组由两个单链RNA区段，所述大（L）（〜7.2 kb的）和小的（S）（〜3.6kb的）段10。而L区段编码的病毒聚合酶（L）和基质蛋白（Z） - 在S段编码病毒核蛋白（NP）和包膜糖蛋白（GP）。的L和S的基因组RNA区段（vRNAs）被打包成病毒颗粒10-12。沙粒病毒感染的最初步骤是病毒颗粒的附着通过病毒GP和其相应的宿主细胞受体的，为JUNV，包括人类的转铁蛋白受体1（hTfR1）13,14之间的相互作用对宿主细胞。以下附件，JUNV颗粒通过网格蛋白介导的内吞作用15取入细胞。颗粒然后投放到晚期内体，其中该隔室的低pH触发的GP 16,17的激活。一旦被激活，在GP-编码的融合肽插入到内体膜，病毒和内体膜之间引发熔合。成功融合结果在释放病毒粒子包装vRNAs的进入细胞质，在那里它们作为用于基因组复制和转录的模板。当产生病毒结构蛋白和vRNAs的足够数量时，新粒子组装和从感染细胞芽完成生命周期18。 为了促进新战略的发现到治疗目标致病沙粒病毒，我们集团一直专注于主机因素是主机的病毒的繁殖至关重要，但可有可无的鉴定。在这种情况下，限定由这种宿主因素控制病毒生命周期的精确阶段（s）是必要的引导的抗病毒策略的发展。在此，我们描述了基于RT-PCR的定量（q）的测定法可用于测量用于识别选定的宿主的蛋白和/或抗病毒分子是否影响病毒进入19的该初始阶段的目的沙粒病毒-细胞附着。衡量沙粒病毒细胞附着替代方法有特色生物素标记20，荧光染料21，或者放射性同位素22的病毒体。缺点这些方法可包括用于大量输入病毒的，使用放射性的（ 例如高感染复数（MOI）的多重），和/或附加的处理步骤以制备输入病毒（ 例如浓缩和/或病毒的标签）的要求。特别是，使用高MOI的使得难以区分生产性与非生产性附着事件15,23。这里描述的基于定量RT-PCR的检测可以用尽可能少的20个牌匾检测附件事件明台病毒（PFU就能），换算成0.0004的MOI为48孔板。因此，测定的强灵敏度克服了高感染复数的要求以及任何病毒标签或浓度的步骤。此外，该测定可以是ⅰ）适于测量病毒体的内吞作用以及病毒基因组的释放入细胞质以下融合和ii）适于通过病毒特异性的qRT-PCR试剂的发展与其他病毒使用（例如，参见24-27）。

<table border="0" cellpadding="0" cellspacing="0" width="730">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">DMEM</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">11965-118</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin-Streptomycin</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">15140-163</td>
			<td style="width:160px;">100x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">HEPES Buffer Solution</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">15630-130</td>
			<td style="width:160px;">100x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">PBS pH 7.4</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">10010-023</td>
			<td style="width:160px;">1x</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Vero E6 cells</td>
			<td style="width:131px;">American Type Culture Collection</td>
			<td style="width:141px;">CRL-1586</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Costar 48-well plate</td>
			<td style="width:131px;">Corning</td>
			<td style="width:141px;">3548</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">RNeasy mini kit</td>
			<td style="width:131px;">Qiagen</td>
			<td style="width:141px;">74106</td>
			<td style="width:160px;">do not mix buffers RLT or&#160; RW1 with bleach</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">QIAshredder homogenizer</td>
			<td style="width:131px;">Qiagen</td>
			<td style="width:141px;">79654</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Taqman Universal PCR Master Mix</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4326614</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Multiscribe Reverse Transcriptase (50U/&#181;l)</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4311235</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">dNTP Mixture (10 mM; 2.5 mM each dNTP)</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">N808-0260</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">GeneAmp 10X PCR Buffer II and 25 mM MgCl2 solution</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">N808-0010</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">RNase Inhibitor (5000U/100 &#181;l)</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">N808-0119</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">RT primer 5&#8217;-AAGGGTTTAAAAATGGTAGCAGAC-3&#8217;</td>
			<td style="width:131px;">Integrated DNA Technologies</td>
			<td style="width:141px;">250 nmol DNA oligo</td>
			<td style="width:160px;">standard desalting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Forward qPCR primer&#160; 5&#8217;-CATGGAGGTCAAACAACTTCCT-3&#8217;</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4304971</td>
			<td style="width:160px;">standard desalting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Reverse qPCR primer 5&#8217;-GCCTCCAGACATGGTTGTGA-3&#8217;</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4304971</td>
			<td style="width:160px;">standard desalting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">TaqMan Probe 5&#8217;-6FAM-ATGTCATCGGATCCTT-MGBNFQ-3&#8217;</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4316033</td>
			<td style="width:160px;">HPLC purified</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">MicroAmp Fast Optical 96-well reaction plate (0.1 mL)</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4346906</td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">StepOnePlus Real-Time PCR System</td>
			<td style="width:131px;">Life Technologies</td>
			<td style="width:141px;">4376598</td>
			<td style="width:160px;">or other qPCR instrument</td>
		</tr>
	</tbody>
</table>

highly sensitive assay for measurement of arenavirus-cell attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

为了证明qPCR分析的灵敏度和动态范围，如在该协议的第4部分中所述编码JUNV C＃1（范围5-5×10 7个拷贝）的NP基因的质粒的已知量经受的qPCR。该测定是敏感的，能够可靠地检测作为目标核酸的5个拷贝（ 图2）少。此外，该测定的动态范围大并且， 如图2，可覆盖至少7日志模板。虽然未示出，我们已检测出核酸的多达5×10 9个拷贝。 为了说明该测定为JUNV C＃1粒子附着的测定的效用，如在该协议中描述的JUNV C＃1各种量被预先结合到Vero E6细胞。洗去未结合的粒子后，将细胞裂解用于RNA纯化和所得的RNA样品进行定量RT-PCR。正如图3所示，测定可以使用少至20个或多达2×10 6 PFU，它转换成的MOI分别为0.0004和40，检测病毒附着事件。 如在图4中所示，可以修改协议不仅测量病毒附着，而且还包含在随着时间的推移输入的病毒颗粒的基因组的扩增的速率。此修改的方法可以用来作为替代，以确定在病毒进入（ 如颗粒的内吞作用和/或融合和病毒基因组的释放入细胞质）的剩余步骤进一步缺陷是否可能发生的。有趣的是，病毒基因组的数量出现的第一个6小时以下的附件，这表明进入的病毒基因组的一部分被降低期间降低。这可能发生在对不被考虑的小区，并降低在细胞外的粒子ular空间或可能由于内溶酶体网络内降解。示出的数据表明，12或18小时后的感染将是最佳的时间来筛选活性基因组复制。 <img alt="图1" src="/files/ftp_upload/53682/53682fig1.jpg" /> 图1:协议工作流的描写的病毒-细胞附着测定法的主要步骤是:1）在4℃与细胞的病毒颗粒的培养，以允许发生随后洗涤病毒-细胞附着以除去未结合的病毒，2）纯化从细胞病毒RNA，3）逆转录（RT），将转换JUNV S片段的基因组RNA（vRNA节）成cDNA，和4）的qPCR来枚举JUNV小号vRNA的副本作为病毒-细胞附着的量度。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53682/53682fig1large.jpg" target="_blank">请按此查看该图的放大版本。</a> 图2:定量PCR法检测JUNV S片段的基因组是敏感的，可以在大的动态范围内使用的JUNV S片段的qPCR引物-探针组是为其精确地测量已知量的能力测试（5，16.6，50， 5×10 3，5×10 5或5×10 7个拷贝）编码所述靶序列的DNA标准对照质粒的。所描绘的扩增曲线（A）和标准曲线拟合线（B）。 R 2，引物探针组的相关系数。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53682/53682fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53682/53682fig3.jpg" /> 图3:JUNV细胞附着测定可靠的措施使用少至20 PFU。Vero E6细胞粒子附着用JUNV C＃已知量1（2×10 1被预先结合，2×10 2 2×10 3，2×10 4，2×10 5个 ，或2 ×10 6 PFU）在4℃下1.5小时。未结合的颗粒通过洗涤除去，将细胞裂解提取RNA，将所得的RNA样品进行定量RT-PCR检测JUNV C＃1 S片段vRNA的作为粒子附着到Vero E6细胞的度量。数据表示，从复孔±标准差每口井的平均拷贝数。 <img alt="图4" src="/files/ftp_upload/53682/53682fig4.jpg" /> 图4:以下感染JUNV C＃1基因组复制的动力学的Vero E6细胞用2×10 3 JUNV的PFU C＃1（0.04 MOI）在4℃下1.5小时温育。以下在冰冷的PBS洗涤几次，将细胞或者立即收获（0小时时间点）或加热到37℃，收集之前指定的时间。在每一种情况下，总RNA从细胞中提取并进行定量RT-PCR检测JUNV C＃1 S片段vRNA节。值列每以及平均拷贝从复孔±SEM。

Watch this Scientific Journal Video about Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment at JoVE.com

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

高灵敏检测方法的沙粒，细胞附着的测量

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

highly-sensitive-assay-for-measurement-of-arenavirus-cell-attachment

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

9.5K 次观看.  University of Vermont. The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

视频: Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation

取得高纯度弓形虫</em一个不连续的氯化铯梯度>卵囊

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

科研

免疫与感染

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2. Manual FV assays have been described 3, 4, but they are time consuming and subjective. Automated FV assays have been reported 5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9. Microplate assays have been reported for clot lysis 10, platelet aggregation 11, and coagulation Factors 12, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma.
This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) 13. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only &mu;l quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity).
Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14. DIC is associated with a poor prognosis and increases mortality above the pre-existing pathology 15. The assay was used to show that in 9 patients with DIC, the FV 1-stage, 2-stage, and total activities were decreased, on average, by 54%, 44%, and 42%, respectively, compared with normal pooled human reference plasma (NHP).
The FV microplate assay is easily adaptable to measure the activity of any coagulation factor. This assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical settings. This information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for coagulation disorders, such as DIC.

This study describes a novel microplate assay that measures FV coagulation activity during fibrin clot formation in human plasma which has not been reported previously. The method uses a kinetic microplate reader to continuously measure the change in absorbance at 405nm during fibrin clot formation in human plasma.

测量人体血浆中的凝血因子V活性，用酶联凝固分析

 In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin ...

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

对小鼠诺沃克类病毒的空斑法

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

定量测定的免疫反应和睡眠模式<em&gt;果蝇</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

生发中心的高度已解决活体条纹照明显微镜

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

人THP-1巨噬细胞通过核转高效转染

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

通过流式细胞仪测量附件和流感的内在病毒在A549细胞

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

对于检测到的敏感性和特异性显色琼脂培养基发展<em&gt;副溶血性弧菌</em&gt;及其他<em&gt;弧菌</em&gt;物种

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

测量<em&gt;体外</em&gt; HIV-1 Preintegration配合物的整合活动

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.