This work was supported by grants from CONACyT-SEP (SEP-2008-84582; CB-2011-01-168497) and Promep-SEP for R.A.G.; P.H. received a scholarship from CONACyT (447442).

1. HFF细胞培养和Ad-感染<ol><li>传播的Ad5 WT病毒在HEK-293细胞和滴度荧光形成单元（FFU）上HFF细胞单层如前面8中所述 。 </li><li>生长人类包皮成纤维细胞（HFF）的10ml DMEM / 10％胎牛血清（FBS）中无菌培养100mm平皿在37ºC和5％ 的 CO 2的加湿培养箱中。确定使用Neubauer室通过在4个16平方集计数细胞的细胞数量。通过计算细胞在4个16平方集的平均数和10 4乘以该数而得到每毫升细胞数，对于包括在步骤1.3每次感染后，用1×10 7 HFF细胞。 </li><li>模拟侵染或感染HFF细胞在用1ml的Ad5的在每个培养皿的DMEM，在30聚焦的MOI 100mm组织培养皿5型腺病毒（Ad5的）野生型（WT）（H5pg4100 8）形成单位，或FFU每个细胞。孵育2小时啊umidified细胞培养培养箱中37ºC和5％的CO 2，小心摇动菜每15分钟，以确保病毒接种物在细胞上均匀分布。在此时间后，除去培养基并加入新鲜的DMEM补充有10％FBS和在湿润的细胞培养培养箱孵育16，24或36小时，在37℃，并 5％的CO 2。继续执行步骤2.1。 </li></ol> 2.制备亚核馏分富含有腺病毒RC <ol><li>收获的Ad5感染或模拟感染HFF细胞与细胞刮收集细胞在无菌离心管。确定细胞数如在步骤1.2。使用1×10 7个细胞用于在步骤1.3中指定的每个时间感染后。 </li><li>离心细胞，在220×g离心，4ºC5分钟。 </li><li>重悬在冰冷的PBS将细胞沉淀（137毫摩尔NaCl，2.7毫米氯化钾，10mM的磷酸氢二钠和1.8mM的KH 2 PO 4）。洗涤细胞沉淀S使用5毫升冰冷PBS每次洗涤3次。为了这个目的，离心细胞，在220×g离心，4ºC5分钟，倾析并弃去上清液（SN），并且重悬细胞沉淀通过温和移液。 </li><li>破坏细胞膜，重悬在700细胞沉淀μ升冰冷的低渗缓冲液（10mM HEPES pH为7.9，10mM的氯化钾，1.5mM的MgCl 2的，0.5mM的DTT，20μ微克/毫升苯甲磺酰氟（PMSF） ，半胱氨酸，丝氨酸，苏氨酸和天冬氨酰蛋白酶抑制剂的混合物包括10μ克/毫升牛胰蛋白酶抑制剂，10μ微克/毫升胃蛋白酶抑制素A和10μ克/ 毫升 N-乙酰基-L-亮氨酰-L-亮氨酰-L- -argininal），并让细胞膨胀在冰上3小时。 </li><li>裂解使用均化器以宽松的A型聚四氟乙烯杵的细胞。执行80 DOUNCE笔画和监视器样品每20笔通过亮视野显微镜，以确保所有细胞已被溶解，但该核具有n个加时赛被损坏或破裂。 </li><li>离心细胞匀浆在300×g离心，4ºC5分钟。存储SN作为在-20 C在离心管中的胞质级分。 </li><li>从细胞核除去细胞碎片，重悬沉淀在750μ升溶液1（S1）的（0.25M蔗糖，10mM的MgCl 2的20μ克/毫升PMSF，半胱氨酸，丝氨酸，的混合物苏氨酸和天冬氨酰蛋白酶抑制剂，包括10 μ克/毫升牛胰蛋白酶抑制剂，10μ微克/毫升胃蛋白酶抑制素A和10μ克/ 毫升 N-乙酰基-L-亮氨酰-L-亮氨酰-L- argininal）和层以上的溶液2等体积（S2） （0.35M的蔗糖，0.5mM的MgCl 2的，20μ克/毫升PMSF，半胱氨酸，丝氨酸，苏氨酸和天冬氨酰蛋白酶抑制剂，包括10μ克/ ml牛胰胰蛋白酶抑制剂，10μ微克/毫升胃蛋白酶抑制素A和10μ的混合物克/ 毫升 N-乙酰基-L-亮氨酰-L-亮氨酰-L- argininal）。 CENTRifuge在1400×g离心，4ºC5分钟。 </li><li>丢弃使用微量的SN。避免干扰颗粒。 </li><li>重悬在含有750μ升S2的分离的细胞核沉淀。 </li><li>为了分离富含腺病毒RC（RCF）亚核分数，超声处理悬浮细胞核用超声波浴，用两个5分钟的脉冲，或直到所有核裂解通过亮视野显微镜观察到。根据需要将样本保持在或低于4℃在超声浴用冰。 </li><li>层的超声处理的核以上的溶液3（S3）（0.88蔗糖，0.5mM的MgCl 2的）和离心机以3,000×g离心，4ºC10分钟的等体积。存放1.5毫升上清液作为核质馏分（NPL）在-70ºC。悬浮颗粒在700μ升S2的和存储的RCF在-70ºC。 </li></ol> RCF 3. Western印迹分析注：对于不良贷款和RCF分数西部印迹分析等预留640 微升的不良贷款和300μl对于RCF从步骤2.11中获得的总体积。 <ol><li>混合15μ升在Laemmli缓冲液2倍每个亚核级分（4％SDS，20％甘油，10％2-巯基乙醇，0.004％溴酚蓝，0.125摩尔Tris盐酸pH6.8）中并煮沸，在95°C下5分钟。加载样品中10％的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS-PAGE）变性凝胶和分离蛋白1.5小时，在20毫安（mA）的。 </li><li>通过以400mA电印迹1.5小时到聚偏二氟乙烯（PVDF）膜转移蛋白。 </li><li>方框膜进行2小时，在使用3％脱脂奶粉的PBS室温。 </li><li>孵育O / N在4ºC与针对病毒的E2A DNA结合蛋白（B6-8 9）第一抗体以1：500稀释在PBS / 0.3％脱脂奶粉。 </li><li>洗涤膜3次，用PBS / 0.1％吐温20 10分钟。 </li><li>孵育小鼠抗IgG二的膜耦合到辣根过氧化物酶（HRP）tibody以1：10,000稀释在PBS中2小时，在室温。 </li><li>用PBS洗膜3次/ 0.1％吐温20 10分钟。 </li><li>开发使用增强的化学发光和X光片的膜。 </li></ol> 4.病毒DNA检测RCF 注：对于从不良贷款和RCF部分DNA的提取，使用210μl对于不良贷款和100μl对于在步骤2.11中获得的总体积的RCF。 <ol><li>在55ºC孵育子核级分1小时用蛋白酶K为1mg / ml和0.5％吐温20。 </li><li>通过温育样品10分钟，在95℃下失活的蛋白酶K。 </li><li>离心样品在20000×g离心，在室温2分钟。 </li><li>收集SN。沉淀DNA与1/10体积的3M醋酸钠和异丙醇中的一个体积，O / N在4℃下。 </li><li>离心样品在20000×g离心，在室温10分钟。 </li><li>丢弃SNü唱微量。在20000×g离心，4℃5分钟洗涤用70％乙醇，离心沉淀。 </li><li>重悬的DNA在10μ升的10mM的Tris-盐酸pH 7.4的</li><li>使用分光光度计通过测量光密度（OD）在260nm处定量的DNA。 </li><li>为了扩增病毒DNA，使用100ng的DNA的使用Taq DNA聚合酶在标准PCR反应各亚核级分在25μ升反应体积的用引物，其允许主要晚期转录单位内的区域的放大，从核苷酸7273至核苷酸7353（81基点）（FW：GAGCGAGGTGTGGGTGAGC; RV：GGATGCGACGACACTGACTTCA）。使用下面的循环条件：初始变性3分钟，在95°C下，之后20个循环的扩增（在95°C下，退火1分钟，在62ºC和延伸30秒，在72ºC1分钟）和用于最终延伸步骤3分钟72ºC。 </li><li>运行PCR产物在2％琼脂糖凝胶上，在90V。色漆乙锭溴化物在0.5μ克/毫升的终浓度和可视化的DNA以证实病毒DNA富集区域合作框架。处理溴化乙锭格外小心，并始终确保要戴手套，因为这种化合物是一种有效的诱变剂。根据制度规定进行处置的溴化乙锭。 </li></ol> 5.晚期病毒mRNA检测的RCF 注：对于从不良贷款和RCF部分RNA分离，用640μl对于不良贷款和300μl对于RCF从步骤2.11中获得的总体积。 <ol><li>使用Trizol根据制造商的说明书分离自亚核级分的RNA。重悬RNA在50μ升DEPC（diethilpyrocarbonate）处理过的水。 </li><li>量化使用分光光度计测量的OD 260nm处的核糖核酸（大约3μg的获得）。存放RNA在50毫微克/ 微升分装置于-70ºC。 </li><li>检查纯化RNA为不存在DNA污染的通过进行在不存在逆转录酶（RT）的控制PCR反应中，用5纳克总RNA，并且在步骤4.9中描述的循环的条件。 <ol><li>如果DNA污染不存在任何扩增子应来制造。如果DNA污染存在，孵育样品与10单位的DNase I U RNA酶抑制剂，10，0.1M的Tris-HCl pH值8.3，0.5M KCl和15mM的MgCl 2的30分钟，在37ºC。继续执行重新分离RNA如在步骤5.1。 </li></ol></li><li>分析相关的RCF病毒mRNA，放大100纳克RNA的通过RT-PCR，使用设计用于检测从不同的基因家族（表1）的腺病毒晚期mRNA的引物各亚核级分。 <ol><li>进行反转录（RT）下，用M-MuLV的逆转录酶在20μ升反应容积为1小时，在42ºC并在70ºC10分钟用100纳克的RNA，来自每个亚核级分在一个标准的RT反应。 </li><li>对于扩增，使用1μ升在PCR反应中，将cDNA的如在步骤4.9。使用下面的循环条件：初始变性3分钟，在95°C下，接着扩增25个循环（在95°C下1分钟，退火在30秒，在72ºC 于表1和延伸规定的温度1分钟）和一为3分钟，在72ºC最终延伸步骤。 </li></ol></li><li>运行RT-PCR产物在2％琼脂糖凝胶上，在90五染色凝胶用溴化乙锭，在0.5μ微克/毫升的最终浓度和可视化，以证实病毒晚期mRNA关联到RCF。处理溴化乙锭如在步骤4.10所示。 </li></ol> RCF 6.免疫可视化注：进位出这个过程在层流柜，以避免样品污染的任何尘埃颗粒，并在使用前过滤所有的解决方案。 <ol><li>现货5 微升步骤2.10可怕获得的RCF分数ctly上的硅烷涂覆的滑动。 </li><li>让光点干约5分钟，在室温。 </li><li>重新水合物缓慢和间接吸取PBS 500μL的RCF点旁边的降低，且让它通过倾斜滑动流动。从侧面流掉过量PBS中。 </li><li>由覆盖用500μ升的PBS / 5％牛血清白蛋白（BSA）的样品进行2小时，在室温块。 </li><li>轻轻和间接吹打的PBS 500μ升到样品洗载玻片3次。倾斜滑动流掉过量PBS中。 </li><li>孵育针对病毒的E2A DNA结合蛋白（B6-8 9）在一个1第一抗体样品1:500稀释在PBS中2小时，在室温。覆盖20μ升抗体溶液的点样的样品，孵育在潮湿室中，以避免干燥。 </li><li>用PBS / 0.02％吐温20轻轻和间接吹打500μ升的PBS到样品洗样品3次。倾斜滑动流掉前塞斯洗涤液。 </li><li>孵育样品与耦合到该被激发在488nm以1荧光团的小鼠抗IgG第二抗体：在4ºC2000稀释在PBS中，1小时。覆盖20μ升抗体溶液的点样的样品，孵育在潮湿室中，以避免干燥。 </li><li>用PBS / 0.02％吐温20轻轻和间接吹打500μ升的PBS到样品洗样品3次。倾斜滑动以排干多余的洗涤溶液中。 </li><li>还包括与使用2μ升的PBS / 10％甘油作为安装平台盖玻片载玻片上的点样的样品。轴封采用透明指甲油和存储人-20ºC。 </li><li>分析使用荧光显微镜的样品中，用63×物镜和一个1.4数值孔径（NA），在488nm处的波长。 </li></ol>

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The authors have nothing to disclose.

In order to elucidate the molecular mechanisms that govern regulation of cellular activities by viral infection understanding the composition and activities associated with RC would be instrumental. Therefore, to make a detailed analysis of RC, we established a cell-free system that takes advantage of the size and biochemical composition of these virus-induced structures, to isolate subnuclear fractions enriched with RC using a simple procedure that relies on velocity gradients with sucrose cushions. Critical steps of th...

腺病毒包含复制在被感染细胞核的双链DNA基因组。当病毒DNA进入细胞核，它局部化相邻的PML核机构1。随着病毒的早期基因的表达，核架构大幅改组，诱导形成病毒性微环境，被称为病毒复制车厢（RC）2。由于腺病毒（广告）RC是网站，病毒基因组复制和病毒晚期基因表达发生，他们提供了一个环境，招募所有参与这些过程中必要的病毒和细胞的因素。有趣的是，各种细胞蛋白负责细胞抗病毒反应，如DNA损伤反应的，先天免疫应答和抑制肿瘤是增选到这些病毒网站2。因此，广告RC可以考虑监管中心之一，促进高效病毒复制，同时伴随调节蜂窝的抗病毒反应，表明这些结构是关键的病毒 - 宿主细胞相互作用的理解。尽管如此，钢筋混凝土形成的分子机制，其组合物和相关活动是知之甚少。 腺病毒的RC，以及RC从复制在核中的其他DNA病毒不相关联的膜，而相比之下，细胞质的RC 3。此外，这些病毒诱导的结构是可能的蛋白质和核酸的待全部由。 RC形成在感染的细胞的RNA病毒（通常被称为病毒工厂）已被分离，同时它们的细胞质定位的和膜结合的状态，这促进了对它们的详细的形态，功能和生化特征4的优势。 据我们所知，核病毒的RC没有被隔离，这可能是由于所述的核内米的核结构和不存在的复杂性embranes这将有利于他们的孤立。他们的研究一直依靠而不是在免疫荧光显微镜，荧光原位杂交和透射电子显微镜。然而，尽管固有的隔离亚核结构复杂，其它核领域，如核仁和Cajal间机构一直在5,6前隔离。因为核仁和RC都是由蛋白质和核酸的，并且具有0.5之间的直径 - 5微米，我们推测，RC也应该适合于隔离。因此，为了更精确地表征相关联的RC的分子组成和功能，我们建立了一种新方法，以分离富含RC亚核级分。为此，我们制备使用速度梯度和蔗糖垫类似程序用于分离核仁7或其他核结构域6子核级分，并建立了无细胞系统，使分子组合物的研究和有关的活动RC。因此这种技术应该提前的病毒 - 宿主细胞相互作用的理解，并表示一个强大的工具，也应有助于RC的详细分析从该复制在核中的其他病毒和诱导形成类似的尺寸的复制隔间的那些形成在腺病毒感染的细胞，如，疱疹病毒，乳头状瘤病毒或多瘤病毒。

<table border="0">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>12100-046</td>
			<td>Warm in 37 &#186;C water bath before use</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>12484-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose, Ultra Pure</td>
			<td>Research Organics</td>
			<td>0928S</td>
			<td>Prepare a 2.55 M stock solution and store at 4 &#186;C</td>
		</tr>
		<tr>
			<td>Dounce homogenizer</td>
			<td>Kontess Glass Company</td>
			<td>884900-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson 1800 Ultrasonic Bath</td>
			<td>Branson</td>
			<td>Z769533&#160;SIGMA</td>
			<td>Turn on 15 min before use.</td>
		</tr>
		<tr>
			<td>Peroxidase AffiniPure F(ab&#39;)&#8322; Fragment Goat Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-036-003</td>
			<td>Use at a 1:10,000 dilution in PBS/0.03% non-fat milk</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG1 Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A-21121</td>
			<td>Use at a 1:2,000 dilution in PBS</td>
		</tr>
		<tr>
			<td>Silane-Prep Slides</td>
			<td>Sigma</td>
			<td>S4651-72EA</td>
			<td>Open in a laminar flow cabinet</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Pierce ThermoScientific</td>
			<td>34080</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of viral replication compartment-enriched sub-nuclear fractions from adenovirus-infected normal human cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

因为病毒复制室（RC）是蛋白质和核酸，类似于其他核结构域组成的亚核病毒诱导的结构，他们证明了通过基于生物化学特征速度梯度以便能够进行隔离。在分馏协议关键步骤示于图1.在每一步骤中，样品需要通过亮视野显微镜进行监测，以确保不同的亚细胞级分的完整性。例如，肿胀的细胞时，需要在低渗缓冲液孵育时间以溶胀细胞质避免了对细胞核进行标准化。细胞均化，完整的?...

Watch this Scientific Journal Video about Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells at JoVE.com

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

由腺病毒感染正常细胞分离病毒复制车厢富集亚核分数

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

isolation-viral-replication-compartment-enriched-sub-nuclear

Research

JoVE Journal

Immunology and Infection

8.7K 次观看.  Universidad Autónoma del Estado de Morelos. We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC. 

视频: Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

富达的RNA病毒和病毒的突变频率的表征的变种的分离

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

科研

免疫与感染

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

从核衣壳的病毒DNA的制备

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

使用Jsubtomo从电子Cryotomography重构平均病毒包膜糖蛋白穗

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

从人类母胎界面分离白细胞

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

从大鼠睾丸支持细胞的分离和管周细胞

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

人雌性生殖道树突状细胞表型鉴定的分离及功能研究

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

人网膜和皮下脂肪组织 CD34+CD31+ 内皮细胞的分离、扩增和脂肪诱导

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...