Name: quantifying human norovirus virus-like particles binding to commensal bacteria using flow cytometry
Uploaded: 2020-04-29T14:00:00
Description: Watch this Scientific Journal Video about Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry at JoVE.com

我们要感谢苏托努卡·巴哈尔和香奈儿·莫斯比-豪德鲁普对书面手稿的批判性审查，以及阿方索·卡里略在生成细菌标准曲线方面的帮助。这项工作部分由国家卫生研究所（R21AI140012）赠款和佛罗里达大学粮食和农业科学研究所的种子赠款资助。

注：协议中概述的细菌生长条件是肠杆菌氯酸和乳酸杆菌的培养条件。为了与其他细菌物种进行病毒：细菌附着测定，所选细菌应在适合细菌的标准条件下培养。
1. 制备细菌生长介质
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肠杆菌生长介质<ol>
<li>通过在1升脱电化（DI）水中溶解10克试油、5克酵母提取物和10克氯化钠（NaCl）来制备液体介质（见材料表）。将所有介质彻底混合...

<ol>
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量化肠道病毒与细菌结合的能力是阐明这些细菌改变病毒感染机制的关键第一步。本文所述的方法经过优化，以测量人类诺如病毒VLP与E.氯环酶（克阴性细菌）和L.gasseri（一克阳性细菌）的相互作用，但可以适应任何哺乳动物病毒和感兴趣的细菌。虽然VLP是活病毒的理想替代品，用于附件测定，这些粒子可以很容易地量化使用流细胞测定，P粒子也被用来检查?...

人诺病毒（HuNOV）是全世界胃肠道疾病的主要原因，每年造成6.85亿例感染和超过20万例死亡。与其他肠道病毒一样，共性细菌的存在已证明能增强该病原体及其代理病毒——鼠诺如病毒22、33的感染。也有相互矛盾的报告，细菌可以抑制感染人类诺如病毒44，5，6。5,6对于几种病毒，病毒和细菌之间的直接相互作用似乎是影响病毒感染的机制的基础22，7，8，9，10，7,8,9,10并且通过电子显微镜证明，人类诺如病毒直接结合到细菌的表面11，12。,12因此，描述这些相互作用对于确定细菌影响病毒感染的机制至关重要。这种特性从量化病毒结合开始，这些细菌物种是宿主微生物群77、12、1312,13的组成部分。这些附件测定不仅揭示了与细菌结合的病毒数量，还有助于确定这种相互作用对病毒健康与生存的影响。
为了量化病毒附着，传统上使用的方法包括基于PCR的检测，量化病毒基因组12或产生放射性标记病毒，并使用闪烁计数来量化病毒颗粒77，8，9，13。8,9,13使用这些方法通常需要获得高数量病毒储存和体外培养技术，从而产生它们。虽然人类诺如病毒的几个培养系统现在存在2,2，14，15，,15没有一个支持所需的强大的复制，以产生这些高度集中的种群，限制或消除使用PCR和闪烁计数来量化人类诺如病毒/细菌相互作用。
为了规避这个问题，病毒状粒子（VlPs）可以作为活病毒的代用品来研究人类诺如病毒和细菌之间的相互作用16，17。16,VLP 是非传染性颗粒，与它们衍生的病毒非常相似。在人类诺如病毒的情况下，这些颗粒是由VP1（以及VP2）蛋白质的表达产生的，这种蛋白质自我组装，创造出缺乏遗传物质（即诺如病毒的RNA）的完整病毒帽。这些VlPs具有很好的特征，在结构和抗原上与野生病毒相似，它们来源于18、19、20、21、22、23。18,19,20,21,22,23因此，VLP是研究人类诺如病毒和共性细菌表面相互作用的理想代用品。鉴于VLP缺乏遗传物质，基于PCR的测定不能用于量化病毒结合。先前曾描述过一种基于抗体的流细胞测定方法，能够以半定量的方式检测与细菌结合的低水平VLP。该方法经过优化，使人类诺如病毒VLP与克阴性和克阳性共性细菌16结合进行精确定量。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5ml Polystrene Round-Bottom Tubes with Cell-Strainer Cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td>After antibody staining, sample are transferred into tubes for flow cytometry analysis.</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma</td>
			<td>A7002</td>
			<td>Used for media preparation</td>
		</tr>
		<tr>
			<td>AnaeroPack</td>
			<td>Thermo Scientific</td>
			<td>R681001</td>
			<td>Anaerobic gas pack used for culture of Lactobacillus gasseri</td>
		</tr>
		<tr>
			<td>BD FacsDiva software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSR Fortessa flow cytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher Bioreagents</td>
			<td>BP1605</td>
			<td>Used for flow cytometry</td>
		</tr>
		<tr>
			<td>Flow Cytometry Stain Buffer (FCSB)</td>
			<td>BD Biosciences</td>
			<td>554657</td>
			<td>Used for flow cytometry</td>
		</tr>
		<tr>
			<td>Mouse IgG2b kappa Isotype Control (eBMG2b), PE, eBioscience</td>
			<td>Thermo Fisher Scientific</td>
			<td>12473281</td>
			<td>Isotype control. This antibody is purchased in the conjugated form from the manufacturer.</td>
		</tr>
		<tr>
			<td>MRS Powder</td>
			<td>BD Biosciences</td>
			<td>288130</td>
			<td>Used for media preparation and to culture Lactobacillus gasseri.</td>
		</tr>
		<tr>
			<td>Norovirus capsid G2 Monoclonal Antibody (L34D)</td>
			<td>Thermo Fisher Scientific</td>
			<td>MA5-18241</td>
			<td>Norovirus GII antibody. This antibody is only available in the unconjugated form and thus must be fluorescently conjugated prior to use in the outlined flow cytometry assays. In this protocol, PE was the chosen fluor, however, other fluorescent molecules can be chosen as best suits the flow cytometer being used by the researcher.</td>
		</tr>
		<tr>
			<td>Norovirus GII.4 VLP</td>
			<td>Creative Biostructure</td>
			<td>CBS-V700</td>
			<td>human norovirus virus like particle, VLPs were generated using the baculovirus system and resuspended in phosphate buffered saline with 10% glycerol. The authors performed independent nanosight tracking analysis to determine the particle concentration of the VLPs. The concentration is approximately 1011 VLPs per milliliter. Based on the protein concentration of the VLPs, approximately 200 particles are added per bacterium in VLP attachment assays.</td>
		</tr>
		<tr>
			<td>PBS 10X</td>
			<td>Fisher Bioreagents</td>
			<td>BP665</td>
			<td>Dilute to 1X prior to use.</td>
		</tr>
		<tr>
			<td>SiteClick R-PE Antibody Labeling Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>S10467</td>
			<td>Conjugation kit used for labling of unconjugated antibody.</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271</td>
			<td>Used for media preparation</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042</td>
			<td>Used for media preparation</td>
		</tr>
		<tr>
			<td>Tube Revolver</td>
			<td>ThermoFisher Scientific</td>
			<td>88881001</td>
			<td>Used in virus:bacterium attachment assay. Set to max speed (40 rpm).</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>BD Biosciences</td>
			<td>212750</td>
			<td>Used for media preparation</td>
		</tr>
	</tbody>
</table>

quantifying human norovirus virus-like particles binding to commensal bacteria using flow cytometry

共生细菌已广为人知，可影响真核病毒的感染。病原体和宿主微生物群之间的直接结合是改变许多这些病毒的感染的原因。因此，描述病毒-细菌结合的性质是阐明细菌改变病毒感染机制所需的基本步骤。对于人类诺如病毒，共性细菌可增强B细胞感染。病毒直接与这些细菌结合，表明这种直接相互作用涉及感染增强机制。多种技术可用于量化细菌和病毒之间的相互作用，包括放射性标记病毒的闪烁计数和聚合酶链反应 （PCR）。这两种方法都需要使用活病毒，可能需要在实验室中生成。目前，人类诺如病毒的成熟体外培养系统都不足以产生高度浓缩的病毒种群。代替活病毒，病毒样颗粒（VLP）被用来描述诺如病毒和细菌之间的相互作用。本文介绍一种流细胞测定方法，使用病毒特异性抗体来量化VLP与克阴性和克阳性细菌结合。只包含细菌和等感控制，可以优化测定，以减少背景抗体结合，并准确量化VLP附件对所测试的细菌。高VLP：细菌比导致VLP与细菌群的较大百分比结合。但是，当 VLP 数量减少时，细菌结合的百分比也会降低。最终，这种方法可用于未来的实验，阐明规范诺如病毒的特定条件和结构成分：细菌相互作用。

图1显示了用于量化人类诺如病毒VLP与交联细菌结合的浇注策略。代表性密度点概述了如何封闭样品以消除细胞碎屑和细胞团块，从而在单体上确定了VLP附件（图1A）。代表直方图显示，细菌中抗诺如病毒抗体信号水平较低，只有缺乏诺如病毒VLP的样品和带有等值对照抗体的VLP细菌样品的低背景信号（<strong class="xfig"...

Watch this Scientific Journal Video about Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry at JoVE.com

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

该协议的目标是量化真核病原体人类诺如病毒与细菌的结合。在进行初始病毒-细菌附件测定后，流细胞测定用于检测种群内的病毒结合细菌。

使用流细胞测定量化人类诺如病毒样颗粒与准细菌结合

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

病毒-细菌相互作用对于成功的病毒感染和刺激宿主免疫反应非常重要。但目前，实验室无法生产高度浓缩和纯化的人类诺如病毒。这种技术可用于任何与细菌结合的病毒，这些病毒可以是病毒样颗粒，也可以是活病毒。它使我们能够量化这些病毒和细菌之间的相互作用。直接从冷冻甘油库存中，用单个分离的肠菌群从果菌块中接种五毫升液体介质。一夜之间长出细菌。第二天，将培养的1.3毫升等分转移到两个独立的1.5毫升离心管中。以10，000 x g离心管5分钟。取出超自然剂，然后每次洗涤使用一毫升无菌 1X PBS 清洗样品两次。再次以10，000 x g将样品离心5分钟。去除上一液，将颗粒重新在1.3毫升无菌1X PBS中。从 0.5 毫升的洗涤培养开始，连续稀释 1X PBS 中的细菌，从 10 到负 1 到 10 到负 4。使用分光光度计测量洗涤未稀释培养物的光学密度，以及 600 纳米的四种稀释光度。对之前的每次稀释执行 10 倍的连续稀释（1 倍 PBS）。将每个系列最后三个稀释的 100 微升扩散到加糖板上，以确定每个样品每毫升形成菌落的单位数。让板在室温下干燥五分钟。倒置盘子，在培养箱中孵育。在准备手稿中描述的试剂、抗体和细菌后，将所有材料组装在 BSL-2 生物安全柜中。人类诺如病毒的病毒类颗粒被列为 BSL-2 病原体，使用 VLP 执行的所有工作都应在经过认证的生物安全柜中进行。将10微克的人类诺如病毒VLPs添加到每根细菌中，通过移液彻底混合。在37摄氏度下，在37摄氏度下孵育管子一小时，并不断旋转。孵育后，以10，000xg离心管5分钟。吸气并丢弃上一升，将细菌颗粒重新在PBS的一毫升中。重复洗涤步骤一次后，将管再次以 10，000 X g 离心五分钟。丢弃上一杯，将VLP细菌颗粒重新在150微升5%阻尼缓冲液中。发生的一个常见错误是交换管子和添加错误的处理。为了防止这种情况，请将所有管在符合正确处理的管内排列，并同时向管中添加一个处理。对于每个细菌样本，制备50微升稀释的人类诺如病毒GII抗体。使用5%的阻尼缓冲液，稀释抗体一至125的E.cloacae样品。使用相同的稀释比，为每个细菌样品制备50微升稀释等型抗体。将每个附件测定样品分成350微升等分，将其转移到干净的1.5毫升离心管中。要创建未污染的控件，请从每个细菌样本的第一个等量中添加 50 微升的阻塞缓冲液。对于染色样品，在第二组等分中加入50微升GII抗体稀释剂。通过将稀释等同型抗体的50微升添加到第三组等同体中，创建等值控制。在冰上和黑暗中孵育所有样品30分钟。然后将所有样品以10，000 X g离心5分钟。丢弃超钠，将每个样品重新丢弃在 FCSB 的 150 微升中。再次，将所有样品以10，000Xg离心5分钟。同样，丢弃超钠，将样品重新在 FCSB 的 100 微升中重新暂停。在最后一次离心样品并丢弃超钠后，将样品重新浸入FCSB的150微升中。将每个样品转移到一个管内，包含 400 微升 FCSB，总体积为 550 微升。将样品储存在4摄氏度，直到通过流式细胞分析。使用流式细胞测量测量了VLP附件。首先，密度图用于清除细胞碎片，然后随后进行点图浇注，以去除细菌双子和细胞团块。代表性直方图显示，与未污染和等型控制相比，仅细菌样品中缺乏PE信号，VLP：细菌样本中PE信号强度发生变化。经过一个小时的孵育与10微克的VLP，流细胞仪检测到粒子结合到E.colacae和L.gasseri。为了确定该测定的结合定量的极限，在加入细菌之前，会生成一个VLPs的稀释序列。VLP 数量的减少导致与受 VLP 约束的细菌百分比相应减少。了解病毒：细菌比并保持实验之间的比率一致，对于解释结果非常重要。细菌和病毒突变体可以产生，以专门确定调解这种相互作用的微生物表面结构。该技术允许研究人员回答有关影响诺如病毒-细菌相互作用的环境和条件的问题，包括病毒基因型的变化、细菌生长条件以及细菌表面结构的变化如何改变病毒结合。

Research

JoVE Journal

Immunology and Infection

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

科研

免疫与感染

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

一种简单有效的方法，通过流式细胞仪检测核转录因子激活人体中性粒细胞

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

造型埃博拉病毒的生命周期在生物安全二级条件下的病毒样颗粒包含Tetracistronic小基因组

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

一种简单快速议定书非酶离解新鲜人组织的浸润淋巴细胞的分析

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

人肺树突状细胞：空间分布与表型鉴定支气管活检采用免疫组化和流式细胞仪

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

使用成像流式细胞仪T细胞同种异体反应性测定

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

影像性流式细胞仪对人体免疫突触的定性和定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

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 ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Preparation of Whole Bone Marrow for Mass Cytometry 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 ...