这项工作是由瑞士国家科学基金会（31003A_135278）海面温度的赠款支持。 MOP是一个博士生津贴从安盛研究基金的受益人。我们感谢帕特里夏·尼格的帮助， 图1的设计。

用活病毒工作时，利用层流罩和适当的生物防护：注意开始之前。在这里，我们描述了适用于文化的流感病毒株A / WSN / 33的生长条件。感染复数（MOI）和温育时间可以根据所用的病毒株而不同。 1.制备生物素标记的A / WSN / 33病毒<ol><li>种子的Madin-Darby犬kindey（MDCK）细胞分化成使用Dulbecco氏改良的Eagle培养基（DMEM），补充有10％FBS和1％青霉素-链霉素（青霉素/链霉素）一个T175 cm 2的烧瓶中。培养细胞在37℃，直到大约70％融合为止。 </li><li>感染前，去除培养基，并通过添加足够大的体积，以覆盖细胞单层洗涤细胞用磷酸盐缓冲盐水（PBS，pH7.0-7.3）。 </li><li>删除PBS和感染细胞用A / WSN / 33与10 -4的MOI。稀释病毒的PBS辅以0.02毫克镁离子，0.01毫克钙&lt;/ SUP&gt;，0.3％牛血清白蛋白（BSA）和1％青霉素/链霉素（感染-PBS）中。使用的4 ml的接种物用于T175 cm 2的烧瓶中。 </li><li>孵育细胞1小时，在37℃。通过吸和吸量管10的10ml DMEM补充有0.3％BSA，20mM的HEPES和1％青霉素/链霉素到烧瓶中除去接种物。 </li><li>孵育细胞，在37℃约3天，直至细胞病变效应（CPE）是可见的，然后收获上清液。 CPE可以被识别为上舍入细胞随后脱离和单元 14的死亡。 </li><li>旋上清液在450×g离心5分钟，以去除细胞碎片。上清转移到一个新的猎鹰管和重复此步骤一次。丢弃粒料。 </li><li>转移上清液入超速离心管和底层上清液用5毫升30％蔗糖的稀释在NTE缓冲液（10mM的Tris [pH为7.4]，100mM NaCl的，​​1mM EDTA）中。超速离心之前平衡管和填充用PBS达到30毫升结束卷。自旋supernatants在112398×g离心（对应于25000转的SW28转子）90分钟在超速离心机。 </li><li>小心地吹打除去上清液，并在250微升PBS浸泡病毒颗粒一夜之间在4℃。悬浮颗粒吹打向上和向下。测量纯化病毒的Bradford试验15蛋白含量。 </li><li>使用生物素化试剂盒来产生生物素化A / WSN / 33。遵循制造商的指令。 </li><li>简言之，添加生物素的适量纯化病毒，并培育2小时在冰上。病毒转移到超速离心机管和填充管与PBS达到30毫升结束卷。旋病毒以112389×g离心在超速离心机90分钟。通过移液除去上清液，加入250微升PBS。颗粒浸泡过夜，在4℃。分装和存储保存在-80℃下的生物素化病毒在那里将是稳定至少24个月。 注意：这可能有助于确定生物素化的p的滴度IAV按标准斑块检测赔偿。但是，应该指出的是，随后的协议只会测量生物素化的病毒颗粒。其余unbiotinylated粒子由于不完全的生物素化将通过噬菌斑测定法，但不要在附接/内化测定法测量的信号向感染滴度。 </li></ol> 2.确定生物素化的病毒将所需量注：在被执行的连接/内化测定法中，以确定生物素化的病毒感染的样品的所有细胞所需的时间量是重要的。 <ol><li>培养，并根据制造商的说明trypsinize A549细胞。计数使用细胞室中的细胞。移液器200000 A549细胞成流式细胞仪深井管。旋3分钟，在450×g下。除去上清液，重悬沉淀用200微升PBS。凉爽的细胞在冰上进行10分钟。 </li><li>制备bioti的5倍连续稀释nylated病毒股票感染PBS。 </li><li>旋管含有细胞在4℃下3分钟，在450×g下。除去上清液，重悬沉淀用100微升含有病毒的感染PBS。由于模拟控制利用感染PBS没有生物素化病毒。冰上孵育管1小时。 </li><li>旋管在4℃下3分钟，在450×g下。在200微升PBS弃上清，重悬沉淀。重复此步骤一次。 </li><li>为固定，旋管在4℃下3分钟，在450×g下。在100微升3.7％多聚甲醛（PFA）的弃上清，重悬沉淀。下室温温育10分钟。然后，重复在2.4中描述的洗涤步骤）。 </li><li>对于透化，旋管在4℃下3分钟，在450×g下。在100微升PBS弃上清，重悬沉淀含有0.5％的Triton X-100。孵育6分钟，在室温。然后，重复在2.4中描述的洗涤步骤）。 </li><li>旋管在4℃下3分钟，在450×g下。除去上清，重悬佩尔等在50μl的2％BSA在PBS中稀释含有STV-Cy3标记。 （请注意，STV-Cy3标记的适当的浓度应在实验开始前测定开始用浓度为1：200和测试2倍稀释到1：2400。在我们手中，稀释为1：1,000证明是最佳的。） </li><li>孵育1小时在黑暗中在室温。然后，在2.4重复洗涤步骤描述，但重悬在200μl2％BSA在PBS中稀释的沉淀。 </li><li>通过流式细胞仪16分析样品。门上的Cy3阳性人群。选择产生的Cy3阳性细胞在A549人口的最大数量的生物素化病毒的最低浓度。 </li></ol> 3.确定链霉的所需量 注：阻断从细胞外病毒导出的信号的效率决定的测定法的检测范围。因此，废除将STV-Cy3的信号尽可能是重要的。钍Ë需要STV的所需量取决于所使用的生物素化的病毒原液（在第2测定）的量。 <ol><li>按照步骤2.1-2.4，但使用在第2测定病毒浓度。 </li><li>制备STV的五倍连续稀释稀释在含有2％BSA和0.1％叠氮化钠的PBS中。 </li><li>旋管在4℃下3分钟，在450×g下。在50微升STV稀释除去上清液，重悬沉淀。由于模拟控制使用含有PBS 2％BSA和0.1％叠氮化钠无STV。孵育管在冰上30分钟。然后，在2.4重复洗涤步骤描述。 </li><li>按照步骤2.5-2.9。用于内化测定法中，选择STV的最低浓度造成的Cy3信号（相对于样品在其中没有STV是本）的一个强烈的块。对于这一点，可以使用CY3阳性细胞或Cy3的信号的平均荧光强度的百分比。理想情况下，STV处理的样品的Cy3的信号应尽可能靠近将信号从模拟感染细胞成为可能。 </li></ol> 4.安装/国际化分析 注：确保在开始试验之前，准备好所有的试剂。对于在结果部分（模拟感染的细胞，神经氨酸苷酶预先处理的细胞和叠氮化钠处理的细胞）呈现三个对照组小改建为协议是必需的。 <ol><li>准备每片含20万的A549细胞16深井管。旁边的实验装置包括4个管中，有3个控制的条件下，每个都需要4个试管：假感染的细胞，神经氨酸酶（NA）-pretreated细胞和叠氮化钠处理的细胞。每个组由4个管标记为“0分钟”，“0分钟+ STV&#39;，&#39;30分钟&#39;，&#39;30分钟+ STV&#39;。 </li><li>旋管在4℃下3分钟，在450×g下。在200微升PBS弃上清，重悬沉淀。 </li><li>旋管在4℃以450×g的。除去上清，重悬佩尔等在200含有0.3％BSA的20mM HEPES和1％青霉素 - 链霉素（孵育培养基）的DMEM微升。育30分钟，在37℃。 注：对于神经氨酸苷酶控制：使用细菌的神经氨酸酶（ 如唾液酸酶来自霍乱弧菌），在200亩/ ml的浓度稀释在孵化培养基悬浮细胞。 </li><li>旋管在4℃下3分钟，在450×g下。在200微升PBS弃上清，重悬沉淀。 10分钟在冰上向下重复此步骤一次，然后冷却试管。 </li><li>旋管在4℃下3分钟，在450×g下。除去上清液，重悬沉淀在100μl生物素化病毒（使用在第2稀释感染PBS确定的病毒浓度）。孵育1小时在冰上。然后，在2.4重复洗涤步骤描述。 注：对于模拟感染控制：使用感染PBS没有病毒。为叠氮化钠控制：使用病毒稀释在感染-PBS中补充有0.1％的叠氮化钠。 </li><li>临cessing在冰上感染后的样品的： <ol><li>对于“0分”样本，按照步骤2.5和存储样品在4°C，直到其他样品是固定的。 </li><li>为&#39;0分钟+ STV的样品，旋管在4℃下3分钟，在450×g下。除去上清，重悬沉淀于50μlSTV（使用第3节中所确定的浓度）稀释在含有2％BSA和0.1％叠氮化钠的PBS（以防止内在化，同时处理的样品）。冰上孵育30分钟。按照步骤2.4-2.5和存储样品在4℃。 </li><li>为&#39;30分钟&#39;的样品，旋管在4℃下3分钟，在450×g下。在200微升含有2％BSA的PBS弃上清，重悬沉淀。孵育30分钟，在37℃。按照步骤2.4-2.5和存储样品在4℃。 注：对于叠氮化钠控制：使用PBS补充有2％BSA和0.1％叠氮化钠的30分钟温育在37℃。 </li><li>为&#39;30分钟+ STV的样品旋管在4℃下3分钟，在450×g下。在200微升含有2％BSA的PBS弃上清，重悬沉淀。孵育30分钟，在37℃。然后，旋管在4℃下3分钟，在450×g下。除去上清，重悬沉淀于50μlSTV（使用第3节中所确定的浓度）稀释在含有2％BSA和0.1％叠氮化钠的PBS（以防止内在化，同时处理的样品）。冰上孵育30分钟。按照步骤2.4-2.5和存储样品在4℃。 注：对于叠氮化钠控制：使用PBS补充有2％BSA和0.1％叠氮化钠的30分钟温育在37℃。 </li></ol></li><li>按照步骤2.6-2.8，并用流式细胞仪分析样品。确定的Cy3阳性细胞的每一个样品中的百分比。 </li><li>计算附着百分比病毒和内病毒的相对量。 <ol><li>要计算附加的病毒的量，使用的Cy3 POSITI的百分比已经门控细胞或Cy3的信号的平均荧光强度。为了比较，设置未处理的细胞为100％（图4B）。 注意：附加的病毒的百分比是由“0分钟”示例说明。 </li><li>为了计算内化的病毒数量，采取的“30分钟+ STV”样本的比例的“30分钟”样品（图5B）。 注意：正如上面提到的，可以使用的Cy3阳性门控细胞或Cy3的信号的平均荧光强度的百分比。 </li></ol></li></ol>

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The authors have nothing to disclose.

我们的协议描述测量病毒的附件和内在化流式细胞仪的一种简单的方法。它允许使用标记的野生型病毒其模拟更加紧密病毒感染相比，使用的病毒样颗粒（VLP）组成。尽管我们的协议进行了优化，以测量附件IAV和内在它可以很容易地适用于其他病毒。此外，如流式细胞术，用于读出，共染色可以很容易地添加到协议如以下击倒的目的蛋白质或过表达来测试表达水平。因此，该测定允许根据?...

A型流感病毒（IAV）的条目是与病毒的受体对靶细胞1的质膜的结合将启动一个多步骤的过程。的受体IAV是唾液酸其存在于大量的各种糖蛋白和糖脂的。 IAV的血凝素（HA）蛋白质，其存在于病毒包膜结合唾液酸，从而介导附着病毒颗粒到靶细胞2的质膜。病毒进入经由网格蛋白介导的内吞作用的细胞，而且还可选条目途径，如巨吞，已经描述3-6。 HA与唾液酸之间的相互作用似乎是足够用于介导两个，附着并触发病毒颗粒7的内化。然而，替代的条目受体已经提出和它们在IAV条目角色目前正在研究1,8,9。 姜黄素rently，均努力确定涉及在病毒的生命周期，与显影急需新的抗病毒疗法10-12中的目的宿主因素。病毒进入将是针对IAV增长，以阻断病毒感染，在其最早的点了有利的一步。测量IAV进入不同阶段的实验是挑战，因为通常大量的病毒都需要检测发来的病毒。此外，检测范围的变化病毒内容只线性由于缺乏病毒复制。这强调了需要检测灵敏度高。 我们在这里介绍的测定法允许附加的病毒的检测在细胞表面以及检测内化病毒的有关细胞相关病毒的总量。使用生物素化的野生型病毒能够通过染色STV-Cy3和读出用流式细胞仪测量方便。生物素化病毒是冷结合到细胞s到允许附件，但阻止病毒颗粒的内化。可将细胞固定，透，并用STV-Cy3的测量附加的病毒。从细胞外，附着病毒体的信号可以如果一个封闭步骤是固定在其中的细胞与非标记的链霉（STV）之前应用废止。在下一步骤中，以下附着生物素化IAV的，温度被转移到37℃和病毒颗粒的内化被允许发生。内化颗粒免受STV结合从而使预算外和细胞内的病毒之间的歧视。 每个实验条件下，四个样品是必需的：“0分钟”：第一个样品，标记为“0分钟”，是在细胞表面以测量冷结合的病毒。 &#39;0分钟+ STV&#39;：第二样品，标为“0分钟+ STV&#39;，给出了实验的基线信号强度。附加的病毒被阻止机智相比于“0分钟”样品H STV和信号下列染色STV-Cy3标记应该是低得多。 &#39;30分钟&#39;：第三样品，&#39;30分钟&#39;包含附着和内在病毒由于温度偏移至37℃30分钟。 &#39;30分钟+ STV“：第四个样品，&#39;30分钟+ STV”措施病毒的细胞内部分。在30分钟培养期之后将STV封闭步骤被应用。其结果是，在细胞表面的病毒颗粒通过STV绑定留下可用于与STV-Cy3的染色只内在病毒。内化病毒的相对量可被计算为内化的病毒（在&#39;30分钟+ STV测定“样品）由病毒的总量（由&#39;30分钟描述划分&#39;样品）的比例。 作为对照，我们建议包括模拟感染的细胞。以下模拟感染的细胞的STV-Cy3的染色的信号给出了背景所得从染色方案。为IAV附件的控制是预处理细胞细菌神经氨酸酶（NA）的。 NA切割开来细胞糖蛋白的唾液酸，从而从细胞表面除去附着受体。叠氮化钠（SA）是一种强效的代谢抑制剂，并由此阻止细胞内吞作用13。用叠氮化钠处理的细胞应该是病毒结合阳性阴性用于内化。

<table border="0" cellpadding="0" cellspacing="0">
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			<td>10270-106</td>
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			<td>VWR Calbiochem</td>
			<td>126579</td>
			<td></td>
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			<td>HEPES</td>
			<td>Life Technologies</td>
			<td>15630-100</td>
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			<td>D-Sucrose</td>
			<td>Fluka</td>
			<td>84100</td>
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			<td>TRIS</td>
			<td>Biosolve BV</td>
			<td>20092391</td>
			<td></td>
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		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>3680</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-LINK NHS-SS-BIOTIN kit</td>
			<td>Fisher Scientific</td>
			<td><a href="https://extranet.fisher.co.uk/insight2_ch/getProduct.do?productCode=W9971E">W9971E&#160;</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Bio Rad Protein Bio Assay</td>
			<td>Bio Rad</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>deepwell tubes (1.2 ml microtubes)</td>
			<td>Milian</td>
			<td>82 00 001</td>
			<td></td>
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			<td>PFA</td>
			<td>&#160;Lucerna chem&#160; Electron microscopy sciences</td>
			<td>15710</td>
			<td></td>
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			<td>ultracentrifuge tubes</td>
			<td>Hemotec HmbH</td>
			<td>253070</td>
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			<td>Triton X-100</td>
			<td>Fluka</td>
			<td>93420</td>
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			<td>STV-Cy3</td>
			<td>Life Technologies</td>
			<td>43-4315</td>
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			<td>STV</td>
			<td>Life Technologies</td>
			<td>43-4302</td>
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			<td>sodium azide</td>
			<td>Fluka</td>
			<td>71290</td>
			<td></td>
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		<tr>
			<td>bacterial neuraminidase/sialidase</td>
			<td>Sigma-Aldrich</td>
			<td>N6514-1UN</td>
			<td></td>
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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.

甲卡通描述四个不同的实验条件下显示在图1代表性实验的结果在图2给出- 。5。在“0分钟”样品，生物素化的病毒是冷结合到靶细胞可以通过STV-Cy3的染色进行可视化（图1和2）。时（在“0分钟+ STV”样品）一个封闭步骤被应用，病毒在细胞表面不能再由STV-Cy3的染色检测。其结果是，在“0分钟+ STV”样品的信号强度被大大...

Watch this Scientific Journal Video about Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry at JoVE.com

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

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 ...

measuring-attachment-internalization-influenza-virus-a549-cells-flow

Research

JoVE Journal

Immunology and Infection

9.9K 次观看.  University of Zurich. 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. 

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

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

在固定和活细胞，流式细胞仪测量钙蛋白酶活性

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

科研

免疫与感染

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

考试流式细胞仪检测胸腺阳性和阴性选择

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

在甲型流感病毒的衣壳的体外拆卸梯度离心

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

流感病毒在小鼠感染模型中的研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

用 MDCK-SIAT1 细胞 Microneutralization 法测定人血清中中和 (H3N2) 病毒的流感中和抗体反应

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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

小鼠与人肺泡巨噬细胞的分离和体外培养

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

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

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

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

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

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

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

利用流细胞测定人类白细胞测定阿斯珀吉鲁斯紫锥体烟曲的噬菌体

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.