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><tbody><tr><td>DMEM</td><td>Life Technologies</td><td>41966-052</td><td></td></tr><tr><td>FBS</td><td>Life Technologies</td><td>10270-106</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies</td><td>15140-163</td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>14190-169&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>BSA</td><td>VWR Calbiochem</td><td>126579</td><td></td></tr><tr><td>HEPES</td><td>Life Technologies</td><td>15630-100</td><td></td></tr><tr><td>D-Sucrose</td><td>Fluka</td><td>84100</td><td></td></tr><tr><td>TRIS</td><td>Biosolve BV</td><td>20092391</td><td></td></tr><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>&lt;a href=&quot;https://extranet.fisher.co.uk/insight2_ch/getProduct.do?productCode=W9971E&quot;&gt;W9971E&amp;nbsp;&lt;/a&gt;</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></tr><tr><td>PFA</td><td>&amp;nbsp;Lucerna chem&amp;nbsp; Electron microscopy sciences</td><td>15710</td><td></td></tr><tr><td>ultracentrifuge tubes</td><td>Hemotec HmbH</td><td>253070</td><td></td></tr><tr><td>Triton X-100</td><td>Fluka</td><td>93420</td><td></td></tr><tr><td>STV-Cy3</td><td>Life Technologies</td><td>43-4315</td><td></td></tr><tr><td>STV</td><td>Life Technologies</td><td>43-4302</td><td></td></tr><tr><td>sodium azide</td><td>Fluka</td><td>71290</td><td></td></tr><tr><td>bacterial neuraminidase/sialidase</td><td>Sigma-Aldrich</td><td>N6514-1UN</td><td></td></tr></tbody></table>

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减少相比...

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

10.1K 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

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

科研

免疫与感染

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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

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

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

病毒融合动力学测量方法在单粒子能级

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

生物学

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.

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 ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

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 ...

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are either time-consuming or unable to detect small variations. Presented here is a protocol for the precise quantification of viral titer by analyzing electrical impedance variations of infected cells in real-time. Cellular impedance is measured through gold microelectrode biosensors located under the cells in microplates, in which magnitude depends on the number of cells as well as their size and shape. This protocol allows real-time analysis of cell proliferation, viability, morphology and migration with enhanced sensitivity. Also provided is an example of a practical application by quantifying the decay of influenza A virus (IAV) submitted to various physicochemical parameters affecting viral infectivity over time (i.e., temperature, salinity, and pH). For such applications, the protocol reduces the workload needed while also generating precise quantification data of infectious virus particles. It allows the comparison of inactivation slopes among different IAV, which reflects their capacity to persist in given environment. This protocol is easy to perform, is highly reproducible, and can be applied to any virus producing cytopathic effects in cell culture.

Reported here is a protocol for the quantification of infectious viral particles using real-time monitoring of electrical impedance of infected cells. A practical application of this method is presented by quantifying influenza A virus decay under different physicochemical parameters mimicking environmental conditions.

使用实时细胞分析监测宿主外的流感病毒存活率

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...

Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

Source: Bailey, M. J., et al. <a href="https://app.jove.com/v/56256">A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies.</a> J. Vis. Exp. (2018).
This video demonstrates an assay measuring the antibody-mediated activation of T cell effector functions. Adding an influenza virus hemagglutinin (HA)-specific monoclonal antibody to transfected mammalian cells expressing HA results in the formation of immune complexes with HA. Upon introducing engineered T cells expressing Fc receptors and a luciferase reporter, the T cells are activated by the antibody-HA complexes, and this activation is detected by adding a luciferase substrate and measuring the produced bioluminescence.

测量 HA 抗体对 Fc 介导的效应子功能的激活

1. Expression of Influenza Virus Hemagglutinin via Transfection

	Plate Human Embryonic Kidney (HEK 293T) cells at a density of 2 x 104 cells/well in a ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

Detection of Bacterial Adherence to Host Cells Using Fluorescence Imaging

Source: Yang, J., et al. <a href="https://app.jove.com/v/62764">Automated, High-Throughput Detection of Bacterial Adherence to Host Cells.</a> J. Vis. Exp. (2021)
This video demonstrates the bacterial adherence assay to evaluate GFP-tagged bacterial adherence to epithelial cell culture to study the dynamics of host-pathogen interactions using fluorescence microscopy-based imaging.

使用荧光成像检测细菌对宿主细胞的粘附

1. Bacterial adherence and host cell staining

	First, wash the seeded A549 cell monolayers three times with warm 1x PBS. For each wash, add 100 &#956;L ...