Name: single cell micro-aspiration as an alternative strategy to fluorescence-activated cell sorting for giant virus mixture separation
Uploaded: 2019-10-27T13:00:00
Description: Watch this Scientific Journal Video about Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation at JoVE.com

作者感谢让-皮埃尔·包多因和奥利维尔·姆巴雷克的建议，并感谢克莱尔·安德烈尼在英语更正和修改方面的帮助。这项工作得到了法国国家研究局根据"投资未来"方案管理，参考ANR-10-IAHU-03（墨西哥感染）和普罗旺斯阿尔卑斯共和国提供的赠款支持。阿祖尔和欧洲基金FEDER PRIMI。

1. 阿米巴文化
<ol><li>使用Vermamoeba 纤维化（应变 CDC19）作为细胞支持。</li>
	<li>在75cm2细胞培养瓶中，在浓度为1 x 106细胞/mL的阿米巴中加入30 mL蛋白酶-肽-酵母提取物-葡萄糖培养基（PYG）和3mL的阿米巴。</li>
	<li>将培养保持在28°C。</li>
	<li>48小时后，使用计数幻灯片对阿米巴进行量化。</li>
	<li>要冲洗，以1 x106细胞/mL的浓度收获细胞，在720 x g下离?...

<ol>
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单细胞微吸气处理的持续时间及其良好功能取决于操作员。实验的不同步骤要求精度。工作站的微操作组件的使用必须通过观察微吸气过程和细胞释放来持续控制。通过微观观察进行随访对于捕获和转移细胞是必要的。有经验的操作员可能需要 1 到 2 小时来分离 10 个细胞，并根据要分离的病毒的丰度逐一重新传输它们。操作的数量可能有所不同。我们建议从 10 个操作开始。根据定义，我们不知?...

核细胞质大DNA病毒（NCLDV）是极其多样化的，由四个家族，感染真核生物1定义。第一个描述的病毒基因组超过300kbp是植物dnavire，包括紫杉醇小球状病毒1 PBCV12。咪咪病毒的分离和首次描述表明，病毒的大小在颗粒大小（450nm）和基因组长度（1.2Mb）3方面都增加了一倍。自那时以来，许多巨大的病毒被描述，通常使用阿米巴共培养程序分离。几个具有不同形态和遗传内容的巨型病毒可以从阿坎塔莫巴病毒细胞中分离出来，包括马赛病毒、潘多拉病毒、皮托病毒、莫利病毒、塞德拉病毒、帕克曼病毒、图潘病毒，以及最近美杜莎病毒4，5，6，7，8，9，10，11，1213，14，15，16，17。同时，对Vermamoeba病毒的分离使得对巨病毒福斯托病毒、考莫埃巴病毒和奥菲奥病毒18、19、20的分离和描述得以进行。其他巨型病毒被分离为他们的宿主原虫，如卡福龙根西21，奥雷球菌阿诺费伦斯22，克雷索穆利纳23和博多盐 24.所有这些隔离都是由于越来越多的团队致力于隔离，并引入了高吞吐量策略更新 25、26、27、28，例如利用流式细胞测定，改进共培养系统。
2016年，我们采用一种将共培养和流细胞学相结合的策略来分离巨型病毒27。制定这一策略是为了增加接种的样本数量，使用作细胞支持的培养者多样化，并快速检测细胞支持的莱沙。该系统通过添加补充步骤来更新，以避免初步的分子生物学鉴定和快速检测未知的病毒群，如Pacman病毒29。耦合流细胞测定细胞分类允许分离咪咪病毒和塞德拉病毒A1130的混合物。然而，我们后来遇到了通过流式细胞测定分离和检测这些病毒亚群的局限性。测序后，当我们组装Fausto病毒ST125和Fausto病毒LCD7（未公布的数据）的基因组时，我们惊奇地发现，每个集合中有两个补充基因组，两个新病毒的基因组在公共基因组数据库中未识别。然而，无论是流动细胞学还是传输电子显微镜（TEM）都显示阿米巴感染了两种不同的病毒，即克兰迪索病毒ST1和佩兰皮塔蒂病毒LCD7。我们设计了特定的PCR系统，分别根据其基因组扩增浮法托病毒、汉迪斯坦病毒和克兰迪索病毒标记;我们的目的是建立基于PCR的系统，能够验证被分离的病毒的纯度。然而，端点稀释和流式细胞测量未能将它们分开。分离这一单一病毒种群是困难的，因为无论是克兰斯蒂诺病毒和汉里帕蒂病毒种群的形态或复制元素都没有特征。由于两个种群的重叠（在有效分离后测试），我们只通过流式细胞测定检测出一个病毒群。我们试图用96孔板的单粒子分拣来分离它们，但我们没有观察到任何细胞病的影响，我们也没有通过PCR扩增来检测克兰斯蒂诺病毒和汉皮拉蒂病毒。最后，只有端点稀释和单阿米巴微吸气的组合，才使得这两种低丰度的巨型病毒从浮力病毒中分离出来。这种分离方法是本文的对象。

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			<td height="21" style="height:21px;width:331px;">Agarose Standard</td>
			<td style="width:185px;">Euromedex</td>
			<td style="width:161px;">Unkown</td>
			<td style="width:391px;">Standard PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">AmpliTaq Gold 360 Master Mix</td>
			<td style="width:185px;">Applied Biosystems</td>
			<td style="width:161px;">4398876</td>
			<td>Standard PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">CellTram 4r Oil</td>
			<td style="width:185px;">Eppendorf</td>
			<td style="width:161px;">5196000030</td>
			<td>Control the cells during the microaspiration process</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Corning cell culture flasks 150 cm2</td>
			<td style="width:185px;">Sigma-aldrich</td>
			<td style="width:161px;">CLS430825</td>
			<td>Culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Corning cell culture flasks 25 cm2</td>
			<td style="width:185px;">Sigma-aldrich</td>
			<td style="width:161px;">CLS430639</td>
			<td>Culture</td>
		</tr>
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			<td height="21" style="height:21px;width:331px;">Corning cell culture flasks 75 cm2</td>
			<td style="width:185px;">Sigma-aldrich</td>
			<td style="width:161px;">CLS430641</td>
			<td>Culture</td>
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			<td height="21" style="height:21px;width:331px;">DFC 425C camera</td>
			<td style="width:185px;">LEICA</td>
			<td style="width:161px;">Unkown</td>
			<td>Observation/Monitoring</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Eclipse TE2000-S Inverted Microscope</td>
			<td style="width:185px;">Nikon</td>
			<td style="width:161px;">Unkown</td>
			<td>Observation/Monitoring</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">EZ1 advanced XL</td>
			<td style="width:185px;">Quiagen</td>
			<td style="width:161px;">9001874</td>
			<td>DNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Glasstic Slide 10 With Counting Grids</td>
			<td style="width:185px;">Kova International</td>
			<td style="width:161px;">87144E</td>
			<td>Cell count</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Mastercycler nexus</td>
			<td style="width:185px;">Eppendorf</td>
			<td style="width:161px;">6331000017</td>
			<td>Standard PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Microcapillary 20 &#181;m</td>
			<td style="width:185px;">Eppendorf</td>
			<td style="width:161px;">5175 107.004</td>
			<td>Microaspiration and release of cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Micromanipulator InjectMan NI2</td>
			<td style="width:185px;">Eppendorf</td>
			<td style="width:161px;">631-0210</td>
			<td>Microcapillary positioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Nuclease-Free Water</td>
			<td style="width:185px;">ThermoFischer</td>
			<td style="width:161px;">AM9920</td>
			<td>Standard PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Optima XPN Ultracentrifuge</td>
			<td style="width:185px;">BECKMAN COULTER</td>
			<td style="width:161px;">A94469</td>
			<td>Virus purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Petri dish 35 mm</td>
			<td style="width:185px;">Ibidi</td>
			<td style="width:161px;">81158</td>
			<td>Culture/observation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Sterile syringe filters 5 &#181;m</td>
			<td style="width:185px;">Sigma-aldrich</td>
			<td style="width:161px;">SLSV025LS</td>
			<td>Filtration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">SYBR green Type I</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:161px;">unknown</td>
			<td>Fluorescent molecular probes/ flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">SYBR Safe</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:161px;">S33102</td>
			<td>Standard PCR; DNA gel stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Tecnai G20</td>
			<td style="width:185px;">FEI</td>
			<td style="width:161px;">Unkown</td>
			<td>Electron microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Type 70 Ti Fixed-Angle Titanium Rotor</td>
			<td style="width:185px;">BECKMAN COULTER</td>
			<td style="width:161px;">337922</td>
			<td>Virus purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ultra-Clear Tube, 25 x 89 mm</td>
			<td style="width:185px;">BECKMAN COULTER</td>
			<td style="width:161px;">344058</td>
			<td>Virus purification</td>
		</tr>
	</tbody>
</table>

single cell micro-aspiration as an alternative strategy to fluorescence-activated cell sorting for giant virus mixture separation

在阿米巴共同培养过程中，多个病毒可能在一个井中分离。我们之前通过应用于病毒群的端点稀释和/或荧光活化细胞分拣 （FACS） 解决了这个问题。然而，当混合物中的病毒具有相似的形态特性，并且其中一种病毒繁殖缓慢时，在基因组组装阶段发现两种病毒的存在，并且病毒无法分离以进一步表征。为了解决这个问题，我们开发了一个单细胞微吸气程序，允许分离和克隆高度相似的病毒。在目前的工作中，我们介绍这种替代策略如何允许我们分离克兰迪索病毒ST1和Usurpati病毒LCD7的小病毒亚群，与溶酶和快速生长相比，这些巨型病毒生长缓慢，不会导致阿米巴溶血症浮病毒通过特定基因扩增来评估纯度控制，并产生病毒以进一步表征。

单细胞微吸气是本手稿中优化的微操作过程（图1）。这种技术能够捕获一个圆的，受感染的阿米巴（图2A），并在含有未感染阿米巴（图2）的新型板中释放它。它是一个适用于共培养系统的功能原型，并成功地分离了非溶性巨病毒。这种方法首次在巨病毒领域被应用，并使得分离出两种新的低丰度巨病毒成为可能。?...

Watch this Scientific Journal Video about Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation at JoVE.com

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

在这里，我们描述了一个单细胞微吸入方法分离受感染的阿米巴。为了分离受福斯托病毒和未知巨病毒感染的Vermamoeba病毒的病毒亚群，我们开发了下面详述的规程，并展示了其分离两种低丰度的新型巨病毒的能力。

单细胞微吸气作为荧光激活细胞分类的替代策略，用于巨型病毒混合物分离

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

单细胞微吸气是一种补充微生物学领域，特别是病毒学领域使用的不同技术的方法。它可以回答关键问题，并解决在巨型病毒分离过程中遇到的问题，将病毒从病毒混合物中分离出，呈现低丰度病毒，因为另一种病毒（即福斯托病毒）的流水线病毒、氏族病毒和乌齐巴蒂病毒的情况是，无论如何，这种病毒都是浮毒病毒。这种方法的主要优点是使用间接战略。它包括分离和克隆受感染的宿主以获得病毒分离。因此，该方法监测从捕获到细胞释放的不同步骤，通过微观观察确认分拣过程。最后，分子生物学用于确认分拣和电子显微镜，以观察分离的病毒颗粒。这种方法将解决病毒分离的主要问题。它是克隆阿梅巴或克隆蛋白的一般前景。首次尝试使用这种技术的人，在控制压力时必须严格而精确。视觉控制对于确认仅捕获和释放一个单元格至关重要。使用VermamoebaVermiformis，菌株CDC19，作为细胞支持。在75平方厘米细胞培养瓶中，以每毫升1倍至6个细胞的浓度加入30毫升PYG培养基和3毫升阿米巴。在孵化器中保持 28 摄氏度的文化。48 小时后，将 10 毫升阿米巴细胞培养转移到计数幻灯片上，将其置于显微镜下以量化阿米巴。要冲洗，在管子中收获30毫升的细胞培养，并在720倍G下离心，在10分钟内对阿米巴进行颗粒状。取出上一代，在适当体积的饥饿介质中重新暂停颗粒，获得每毫升10倍到第六细胞的浓度。准备细胞支持与添加抗菌剂。在生物安全柜中，将阿米巴培养物的十倍加一十，加入两毫升的饥饿介质中。然后接种含有病毒混合物的100毫升的库存溶液，以0.01的多重感染将细胞培养支持接种。孵育受感染的阿米巴细胞培养支持。加入30摄氏度10至14小时或直到细胞病变效应诱导，如阿米巴四舍五入或解解。接下来，使用注射器收集介质并通过五微米过滤器过滤，以清除细胞碎片。在不同的井中对病毒样本进行谷类稀释。接种两毫升的一倍10至第六Vermamoba Vermaformis包含在每个培养皿与100毫升的混合物接种。然后，在30摄氏度下将培养皿放入可密封的塑料袋中。感染后6小时，用倒置光学显微镜观察培养皿，每四到八小时检查一次细胞形态。抗微生物治疗用于细胞支持。在培养皿中选择那些由真菌和细菌剂造成的视觉污染，以及由于病毒和阿米巴的预解和舍入阶段对阿米巴的细胞病变作用的证据，以避免病毒颗粒的吸入。设置带微型机械手、手动控制压力装置、倒置显微镜、即插即用电机模块、摄像头和计算机模块的工作站。选择 20 微微毛细管内径，以便维护内部位置和轻松释放电池。将电动模块上夹持系统的操作角度固定 45 度。执行双重安装，首先在抓握系统，然后对微型毛细管。专注于细胞。要克隆细胞，请将含有两毫升受感染阿米巴的培养皿放在显微镜下。首先关注细胞，然后关注浸入培养中的微毛细管。挑选圆形单细胞，使微毛细管更接近微操纵器。通过手动压力控制，对细胞进行手动压力控制，将单个细胞从第一个样本中取出并释放到阿米巴的细胞培养支持中。然后在30摄氏度下孵育。仔细控制压力，以便能够渴望一个细胞，并控制你的愿望，通过观察单个细胞的释放。使用倒置光学显微镜进行日常观察，以观察细胞的外观并监测细胞病变效应的出现。现在运行一个自动提取系统，从部分正培养样本中提取DNA，其中观察到细胞病变效应。设计底像，用于放大被注释为福斯托病毒的 RBP2 的不良基因、Usurpat 病毒的主要帽状蛋白和 Clandestino 病毒的轻微帽状蛋白。根据手稿使用热循环器执行标准 PCR。执行 20 微升的 PCR 反应，每个引水 50 微摩尔，1X 主混合，和核糖核酸酶无水。在135伏特电压下，在1.5%的加糖凝胶上用DNA凝胶染色运行PCR产品，并用紫外线进行可视化。该协议优化了具有单细胞微吸气的微管理过程。这种技术能够捕获一个圆形和受感染的阿米巴，并在一个包含未感染的阿米巴的新盘子里释放。这种方法成功地分离出一种名为 Userpat病毒LCD7 的新低丰度巨型病毒。您的用户帕特病毒仅在克隆七中观察到，存在 Userpatvirus LCD7 DNA 和福斯托病毒 DNA。电子显微镜揭示了克兰德斯蒂诺病毒ST1的外观，它有一个典型的无纤维的异体形态学以及具有约250纳米的宇宙膜帽的乌苏尔帕特病毒LCD7。低细胞学证实浮士德病毒和纳沃伊鲁病毒种群的重叠。一旦掌握了，这个程序可以很容易地完成。尝试这一程序时，要记住的最重要的事情是有必要对实体的规模进行最低限度的观察仪式，对工作站的主管进行良好处理，并检查任何污染问题。该方法可作为基于细胞形态的排序技术。目前，它用于我们的实验室克隆特定的阿米巴。这项技术的一个关键点是保持细胞的完整性。事实上，我们可以有一些问题，不观察细胞或释放。这主要是因为细胞分解成微毛细管。当缺陷遥测和事实排序不可用以及达到特定技术限制时，此方法可能是有效的替代方案。不要忘记，在某些情况下，细胞不会从微毛细管中释放。这部分是由于阿米巴的特性及其适应能力，然后从细胞膜。

Research

JoVE Journal

Biology

Fluorescence Activated Cell Sorting of Plant Protoplasts

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

科研

生物学

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

溴脱氧尿嘧啶核苷（BrdU）标记和文化独立的鉴定溶解有机随后的荧光激活细胞分选碳有辱人格的浮游细菌

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

琼脂糖凝胶电泳分离DNA片段

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

细胞特异性分析<em&gt;拟南芥</em&gt;叶用荧光激活细胞分选

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

新鲜组织分离正常和癌症相关的成纤维细胞的荧光激活细胞分选（FACS）

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Single-cell Microinjection for Cell Communication Analysis

Cell的传播分析单细胞显微注射

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

敏感，大规模定量代谢组学的策略

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

对于样品制备与TEM分析低细胞数量的另一种方法

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

荧光激活细胞分拣，用于分离瘦杆菌细胞群

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...