Name: automated separation of c. elegans variably colonized by a bacterial pathogen
Uploaded: 2014-03-21T15:00:00
Description: Watch this Scientific Journal Video about Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen at JoVE.com

作者感谢埃利森医学基金会的支持。我们还要感谢艾比Dernburg实验室的成员，使用的wormsorter援助。

1。获得年轻的成年动物的同步文化<ol><li>蠕虫生长在接种OP50-1 大肠杆菌数NGM板大肠杆菌菌（10倍浓缩从饱和的文化），直到许多蠕虫都达到了妊娠阶段。 </li><li>治疗妊娠动物的卵制备的溶液，以获得同步培养（卵）。 </li><li>在几个60毫米NGM板板蛋接种10倍浓缩OP50-1在大约150-200个卵/板的密度。 </li><li>孵育板在25°C为2天（直到蠕虫已经达到了L4 - 青少年阶段）。 </li></ol><p class="jove_ti...

<ol>
	<li>Pulak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+analysis,+sorting,+and+dispensing+of+C.+elegans+on+the+COPAS+flow-sorting+system.">Techniques for analysis, sorting, and dispensing of C. elegans on the COPAS flow-sorting system.</a> Methods Mol. Biol. , 275-286 (2006).</li><li>Dupuy, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+analysis+of+in+vivo+spatiotemporal+promoteractivity+in+Caenorhabditis+elegans.">Genome-scale analysis of in vivo spatiotemporal promoteractivity in Caenorhabditis elegans.</a> Nat. Biotechnol. 25, 663-668 (2007).</li><li>Squiban, B., Belougne, J., Ewbank, J., Zugasti, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+Automated+High-throughput+Genome-wide+RNAi+Screens+in+elegans.">Quantitative and Automated High-throughput Genome-wide RNAi Screens in elegans.</a> J. Vis. Exp. (60), (2012).</li><li>Doitsidou, M., Flames, N., Lee, A. C., Boyanov, A., Hobert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+screening+for+mutants+affecting+dopaminergic-neuron+specification+in+C.+elegans.">Automated screening for mutants affecting dopaminergic-neuron specification in C. elegans.</a> Nat. Methods. 5, 869-872 (2008).</li><li>Pujol, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+innate+immune+responses+to+infection+and+wounding+in+the+C.+elegans+epidermis.">Distinct innate immune responses to infection and wounding in the C. elegans epidermis.</a> Curr. Biol. 18, 481-489 (2008).</li><li>Medzhitov, R., Janeway, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+recognition:+mechanisms+and+pathways.">Innate immune recognition: mechanisms and pathways.</a> Immunol. Rev. 173, 89-97 (2000).</li><li>Matzinger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance,+danger,+and+the+extended+family.">Tolerance, danger, and the extended family.</a> Annu. Rev. Immunol. 12, 991-1045 (1994).</li><li>Mariathasan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopyrin+activates+the+inflammasome+in+response+to+toxins+and+ATP.">Cryopyrin activates the inflammasome in response to toxins and ATP.</a> Nature. 440, 228-232 (2006).</li><li>Melo, J. A., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+conserved+C.+elegans+genes+engages+pathogen-+and+xenobiotic-associated+defenses.">Inactivation of conserved C. elegans genes engages pathogen- and xenobiotic-associated defenses.</a> Cell. 149, 452-466 (2012).</li><li>Chen, G. Y., Nunez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterile+inflammation:+sensing+and+reacting+to+damage.">Sterile inflammation: sensing and reacting to damage.</a> Nat. Rev. Immunol. 10, 826-837 (2010).</li><li>Tenor, J. L., Aballay, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+Toll-like+receptor+is+required+for+Caenorhabditis+elegans+innate+immunity.">A conserved Toll-like receptor is required for Caenorhabditis elegans innate immunity.</a> EMBO Rep. 9, 103-109 (2008).</li><li>Pujol, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reverse+genetic+analysis+of+components+of+the+Toll+signaling+pathway+in+Caenorhabditis+elegans.">A reverse genetic analysis of components of the Toll signaling pathway in Caenorhabditis elegans.</a> Curr. Biol. 11, 809-821 (2001).</li><li>McEwan, D. L., Kirienko, N. V., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+translational+inhibition+by+Pseudomonas+aeruginosa+Exotoxin+A+Triggers+an+immune+response+in+Caenorhabditis+elegans.">Host translational inhibition by Pseudomonas aeruginosa Exotoxin A Triggers an immune response in Caenorhabditis elegans.</a> Cell Host Microbe. 11, 364-374 (2012).</li><li>Dunbar, T. L., Yan, Z., Balla, K. M., Smelkinson, M. G., Troemel, E. R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+detects+pathogen-induced+translational+inhibition+to+activate+immune+signaling.">C. elegans detects pathogen-induced translational inhibition to activate immune signaling.</a> Cell Host Microbe. 11, 375-386 (2012).</li><li>Irazoqui, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+pathogenesis+and+host+responses+during+infection+of+C.+elegans+by+P.+aeruginosa+and+S.+aureus.">Distinct pathogenesis and host responses during infection of C. elegans by P. aeruginosa and S. aureus.</a> PLoS Pathog. 6, (2010).</li><li>Pukkila-Worley, R., Ausubel, F. M., Mylonakis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+infection+of+Caenorhabditis+elegans+induces+antifungal+immune+defenses.">Candida albicans infection of Caenorhabditis elegans induces antifungal immune defenses.</a> PLoS Pathog. , (2011).</li><li>Shapira, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+role+for+a+GATA+transcription+factor+in+regulating+epithelial+innate+immune+responses.">A conserved role for a GATA transcription factor in regulating epithelial innate immune responses.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 14086-14091 (2006).</li><li>Troemel, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p38+MAPK+regulates+expression+of+immune+response+genes+and+contributes+to+longevity+in+C.+elegans.">p38 MAPK regulates expression of immune response genes and contributes to longevity in C. elegans.</a> PLoS Genet. , 183 (2006).</li><li>Twumasi-Boateng, K., Shapira, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissociation+of+immune+responses+from+pathogen+colonization+supports+pattern+recognition+in+C.+elegans.">Dissociation of immune responses from pathogen colonization supports pattern recognition in C. elegans.</a> PLoS One. , 7 (2012).</li><li>Jakobsen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+alkaloid+compound+harmane+increases+the+lifespan+of+Caenorhabditis+elegans+during+bacterial+infection,+by+modulating+the+nematode's+innate+immune+response.">The alkaloid compound harmane increases the lifespan of Caenorhabditis elegans during bacterial infection, by modulating the nematode's innate immune response.</a> PLoS One. 8, (2013).</li><li>Portal-Celhay, C., Bradley, E. R., Blaser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+intestinal+bacterial+proliferation+in+regulation+of+lifespan+in+Caenorhabditis+elegans.">Control of intestinal bacterial proliferation in regulation of lifespan in Caenorhabditis elegans.</a> BMC Microbiol. 12, 49 (2012).</li><li>Troemel, E. R., Felix, M. A., Whiteman, N. K., Barriere, A., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsporidia+are+natural+intracellular+parasites+of+the+nematode+Caenorhabditis+elegans.">Microsporidia are natural intracellular parasites of the nematode Caenorhabditis elegans.</a> PLoS Biol. 6, 2736-2752 (2008).</li><li>Montalvo-Katz, S., Huang, H., Appel, M. D., Berg, M., Shapira, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+with+soil+bacteria+enhances+p38-dependent+infection+resistance+in+Caenorhabditis+elegans.">Association with soil bacteria enhances p38-dependent infection resistance in Caenorhabditis elegans.</a> Infect. Immun. 18, 514-520 (2013).</li></ol>

我们所描述的方法利用实体的荧光标记的蜗杆以外，遵循蠕虫和它的环境之间的相互作用。在我们目前的情况下，分离是基于病原体的标记和被雇用，以单独的蠕虫与那些没有（或轻）负荷沉重的病原体负荷。随后的基因表达分析中发现的免疫反应，两组表明它们是独立的病原体负载之间没有差异。启动该响应信号别处被证明在物理上与细菌相关的，而不是被分泌的，这表明这种信号可以是PAMP。...

蠕虫自动分拣很像流式细胞仪，通过在蠕虫（通常由记者蛋白的转基因表达提供）测量荧光信号，因为它通过拉直在管经营，允许根据重定向到任何一个收集管/孔或废物容器到门的研究员1设置参数。该wormsorter可以在许多方面促进研究，采用它作为分析工具的一个例子是一项研究，随后启动子活性的时空模式对近1000个基因2。 然而，wormsorter的主要用途是在遗传筛选，下列靶基因表达水平或荧光蛋白的沿蜗杆3-5的轴线定位。 在这里，我们描述了wormsorter一个新的应用程序，在由荧光标记的病原体以下蠕虫的殖民化。用此作为一种工具，我们专注于pathog之间的关系恩定植/负载和免疫反应，从而获得新的见解负责的蠕虫启动免疫反应的机制。 几乎在研究迄今所有的生物，先天免疫反应，病原微生物开始依赖于识别病原体相关分子模式（PAMP），和/或危险/损伤相关分子模式（抑）6,7。第一是保守的微生物结构，其包括微生物细胞壁，其鞭毛，或它的脂质双分子层6的部件，第二个，包括释放分子（ 如 ATP 8），改变的蛋白或改变的细胞过程9,10的其他标记。这两种类型的信号被指定为模式识别受体（模式识别受体），其在特定的图案分子的结合激活链导致的保护性反应事件的蛋白C。确认。 线虫一直作为tracta非常有用的BLE模型解剖的宿主 - 病原体相互作用的各个方面，但有一点还不是很清楚的是免疫应答是如何在蠕虫发起的。无推定的受体是同源模式识别受体（的PRRs）在其他生物体中已显示出结合的PAMP，许多的PRR是关键对于在其他生物中的免疫应答的直向同源物的展现给蠕虫病原体响应一个令人惊讶的贡献有限和电阻。例如，在果蝇 Toll受体，它是一种抗革兰氏阳性病原体必不可少的，表示以C由一个唯一同源物，TOL-1，这有助于保护从革兰氏阴性病原体沙门氏菌 11，但不从其他检测革兰氏阴性或阳性病原体11,12。这些观察，结合数据线虫表明免疫应答可以通过破坏细胞蛋白质翻译h内诱导为使一些人认为，C。线虫主要检测DAMPS 9,13,14。然而，描述的死病原体诱导的免疫应答能力的报告表明，PAMP结合可能有病原体识别在C的重要作用线虫 15,16。以前的工作重点是与年龄同步的基因完全相同C.免疫反应线虫种群，由细菌革兰氏阴性致病菌绿脓杆菌表现在肠道定植大的个体差异。 然而，转录谱研究处理这些可变定植人群作为一个实体17,18。利用这种变化，我们开发了一套集中的自动化wormsorter分离暴露在表达GFP的体育秀丽隐杆线虫的差异殖民地人口铜绿假单胞菌 。研究基因表达的不同定植人群FACI病原体负载（和相关联的损伤）和免疫应答和有关C.病原体识别提供了新的见解之间的关系litated评估线虫 19。下面我们介绍的协议，它可以应用到排序感染任何荧光标记的病原虫。 给潜在的用户应当注意的是，需要进行排序的蠕虫数目依赖于随后的分析和协议中使用的性质。例如，在微阵列基因表达分析的情况下，&gt; 1,000蠕虫将需要获得足够的RNA，如果使用标准的协议，但〜100蠕虫就足够了，如果扩增采用，允许快速采集材料，因而应力最小化，以该蠕虫。

<table border="0" cellpadding="0" cellspacing="0" width="927">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">M9 Buffer</td>
			<td style="width:139px;">Prepared in house</td>
			<td style="width:527px;">Recipe at wormbook.org</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rifampicin&#160;</td>
			<td>Sigma</td>
			<td>R3501</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Egg prep solution&#160;</td>
			<td>Prepared in house</td>
			<td>50ml water ; 40ml bleach ; 10ml of 10N Sodium Hydroxide&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NGM plates&#160;</td>
			<td>Prepared in house</td>
			<td>Recipe at wormbook.org</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SKP plates</td>
			<td>Prepared in house</td>
			<td>Recipe same as NGM only 0.35% peptone instead of 0.25%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Control test particles&#160;</td>
			<td>Union Biometrica&#160;</td>
			<td>310-5071-001</td>
		</tr>
	</tbody>
</table>

automated separation of c. elegans variably colonized by a bacterial pathogen

该wormsorter是一种手段类似于用在秀丽隐杆线虫的研究，在FACS机器，通常进行排序基于荧光报道的表达蠕虫。在这里，我们重点介绍该仪器的另一种用法，根据由GFP表达他们的病原菌定植程度的排序蠕虫。这个新的使用使我们能够解决肠蠕虫和诱导的免疫应答的定植之间的关系。虽然C.线虫的免疫反应，以不同的病原体已被记录在案，仍是一个未知数，什么因素促使他们。两个主要的可能性（这并不相互排斥）是识别病原体相关的分子模式，并检测感染所引起的损伤。要在两个可能性区分，接触到的病原体必须从它所造成的损害进行分离。蠕虫的wormsorter启用分离的进行了广泛定植由革兰氏-Negative致病菌绿脓杆菌 ，有可能是由病原体负荷造成的损害，从蠕虫，亦放置，而不是，或轻微，定植。这些不同的群体被用来评估病原体负载和转录的免疫应答的诱导之间的关系。该结果表明，这两个解离，配套的病原体识别的可能性。

当年龄相匹配的，遗传上相同的C。线虫暴露于P。假单胞菌 ，广泛分布在定殖（ 图1A）的水平变化。与此处描述的noncolonized从殖民蠕虫有效的分离可以实现（ 图1B）协议的帮助。不像noncolonized蠕虫，蠕虫殖民显示损坏的迹象，如呆滞，减少排便19。后者可能是一个原因，蠕虫殖民地P。绿脓杆菌有困难清除感染。这对描述的协议表明蠕虫排序，n...

Watch this Scientific Journal Video about Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen at JoVE.com

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

自动分离<em&gt; C。线虫</em&gt;可多段由细菌病原体殖民者

该wormsorter通过根据荧光记者表达分拣蠕虫有利于遗传筛选在秀丽隐杆线虫 。在这里，我们描述了一个新的使用：根据定植通过GFP表达病原体排序，我们用它来检查病原体识别在启动免疫反应的了解甚少的作用。

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

Research

JoVE Journal

Immunology and Infection

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

Invasion of Human Cells by a Bacterial Pathogen

由细菌病原体入侵人体细胞

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

科研

免疫与感染

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

为期一天的血培养的细菌病原体检测和耐药性检测的工作流程计划

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

成像InlC的分泌由细菌病原体调查感染蜂窝<em&gt;单核细胞增生李斯特氏菌</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

从非耕种植物病原菌利用酵母双杂交筛选Unravelling一个细菌效应中的作用

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

质谱法富集细菌脂蛋白及 n-端脂肽的制备结构测定

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

一种高通量、高含量、液态的 Pathosystem线虫

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

利用广域跟踪平台对大型线虫种群进行自动行为分析

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

细菌病原体在细胞和有机水平宿主反应探针中的应用

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...