Name: application of flow vermimetry for quantification and analysis of the caenorhabditis elegans gut microbiome
Uploaded: 2023-03-31T14:20:00
Description: Watch this Scientific Journal Video about Application of Flow Vermimetry for Quantification and Analysis of the Caenorhabditis elegans Gut Microbiome at JoVE.com

这项工作得到了美国国立卫生研究院（NIH）资助DP2DK116645（BSS），邓恩基金会飞行员奖和NASA资助80NSSC22K0250（BSS）的支持。该项目还得到了贝勒医学院细胞术和细胞分选核心的支持，并获得了 CPRIT 核心设施支持奖 （CPRIT-RP180672）、NIH（S10 OD025251、CA125123 和 RR024574）的资助，以及 Joel M. Sederstrom 的协助，以及 LPS NIH 资助的仪器资助（S10 OD025251）。一些菌株由CGC提供，CGC由NIH研究基础设施计划办公室（P40 OD010440）资助。

1.微生物组混合物的制备
<ol><li>将甘油冷冻原液中的细菌压印或划出到溶原肉汤（LB）平板或适当的生长培养基上，并在基于目标细菌菌株的最佳温度下生长过夜（ 秀丽隐杆线 虫天然微生物通常为25°C）。</li>
	<li>从LB板中，使用来自每个细菌分离株的单个菌落（例如，CeMbio系列的12个细菌）在1mL深孔板的单独孔中接种800μLLB培养基。在最佳温度下孵育过夜，以 250 rpm 振荡。 ?...

<ol>
	<li>McFall-Ngai, 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=Animals+in+a+bacterial+world,+a+new+imperative+for+the+life+sciences.">Animals in a bacterial world, a new imperative for the life sciences.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (9), 3229-3236 (2013).</li><li>Seedorf, 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=Bacteria+from+diverse+habitats+colonize+and+compete+in+the+mouse+gut.">Bacteria from diverse habitats colonize and compete in the mouse gut.</a> Cell. 159 (2), 253-266 (2014).</li><li>B&#228;ckhed, F., 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+gut+microbiota+as+an+environmental+factor+that+regulates+fat+storage.">The gut microbiota as an environmental factor that regulates fat storage.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (44), 15718-15723 (2004).</li><li>Turnbaugh, P. J., 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=An+obesity-associated+gut+microbiome+with+increased+capacity+for+energy+harvest.">An obesity-associated gut microbiome with increased capacity for energy harvest.</a> Nature. 444 (7122), 1027-1031 (2006).</li><li>Gill, S. 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=Metagenomic+analysis+of+the+human+distal+gut+microbiome.">Metagenomic analysis of the human distal gut microbiome.</a> Science. 312 (5778), 1355-1359 (2006).</li><li>Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensation+in+C.+elegans.">Chemosensation in C. elegans.</a> WormBook: The Online Review of C. elegans Biology. , 1-29 (2006).</li><li>Couillault, C., 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=TLR-independent+control+of+innate+immunity+in+Caenorhabditis+elegans.+by+the+TIR+domain+adaptor+protein+TIR-1,+an+ortholog+of+human+SARM.">TLR-independent control of innate immunity in Caenorhabditis elegans. by the TIR domain adaptor protein TIR-1, an ortholog of human SARM.</a> Nature Immunology. 5 (5), 488-494 (2004).</li><li>Kim, D. 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=A+conserved+p38+MAP+kinase+pathway+in+Caenorhabditis+elegans+innate+immunity.">A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity.</a> Science. 297 (5581), 623-626 (2002).</li><li>Berg, 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=TGF&#946;/BMP+immune+signaling+affects+abundance+and+function+of+C.+elegans+gut+commensals.">TGF&#946;/BMP immune signaling affects abundance and function of C. elegans gut commensals.</a> Nature Communications. 10 (1), 1-12 (2019).</li><li>Garsin, D. A., 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=Long-lived+C.+elegansdaf-2+mutants+are+resistant+to+bacterial+pathogens.">Long-lived C. elegansdaf-2 mutants are resistant to bacterial pathogens.</a> Science. 300 (5627), 1921 (2003).</li><li>Cabreiro, F., Gems, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worms+need+microbes+too:+microbiota,+health+and+aging+in+Caenorhabditis+elegans.">Worms need microbes too: microbiota, health and aging in Caenorhabditis elegans.</a> EMBO Molecular Medicine. 5 (9), 1300-1310 (2013).</li><li>Samuel, B. S., Rowedder, H., Braendle, C., F&#233;lix, M. -. 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=Caenorhabditis+elegans+responses+to+bacteria+from+its+natural+habitats.">Caenorhabditis elegans responses to bacteria from its natural habitats.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (27), 3941-3949 (2016).</li><li>Dirksen, P., 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+native+microbiome+of+the+nematode+Caenorhabditis+elegans:+gateway+to+a+new+host-microbiome+model.">The native microbiome of the nematode Caenorhabditis elegans: gateway to a new host-microbiome model.</a> BMC Biology. 14 (1), 38 (2016).</li><li>Berg, 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=Assembly+of+the+Caenorhabditis+elegans+gut+microbiota+from+diverse+soil+microbial+environments.">Assembly of the Caenorhabditis elegans gut microbiota from diverse soil microbial environments.</a> The ISME Journal. 10 (8), 1998-2009 (2016).</li><li>Zhang, F., 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=Caenorhabditis+elegans+as+a+model+for+microbiome+research.">Caenorhabditis elegans as a model for microbiome research.</a> Frontiers in Microbiology. 8, 485 (2017).</li><li>Zhang, F., 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=Natural+genetic+variation+drives+microbiome+selection+in+the+Caenorhabditis+elegans+gut.">Natural genetic variation drives microbiome selection in the Caenorhabditis elegans gut.</a> Current biology: CB. 31 (12), 2603-2618 (2021).</li><li>Dirksen, P., 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=CeMbio+-+The+Caenorhabditis+elegans+microbiome+resource.">CeMbio - The Caenorhabditis elegans microbiome resource.</a> G3. 10 (9), 3025-3039 (2020).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook: The Online Review of C. elegans Biology. , 1-11 (2006).</li><li>R Core. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Team+R:+A+language+and+environment+for+statistical+computing.">Team R: A language and environment for statistical computing.</a> R Core. , (2018).</li><li>Wickham, 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=.">.</a> tidyverse. , (2019).</li><li>Anderson, Q. L., Revtovich, A. V., Kirienko, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput,+high-content,+liquid-based+C.+elegans+pathosystem.">A high-throughput, high-content, liquid-based C. elegans pathosystem.</a> Journal of Visualized Experiments. (137), e58068 (2018).</li><li>Twumasi-Boateng, K., 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=Automated+separation+of+C.+elegans+variably+colonized+by+a+bacterial+pathogen.">Automated separation of C. elegans variably colonized by a bacterial pathogen.</a> Journal of Visualized Experiments. (85), e51090 (2014).</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 Microbiology. 12 (1), 49 (2012).</li><li>Zhang, F., 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=High-Throughput+assessment+of+changes+in+the+Caenorhabditis+elegans+gut+microbiome.">High-Throughput assessment of changes in the Caenorhabditis elegans gut microbiome.</a> Aging: Methods and Protocols. 144, 131-144 (2020).</li><li>Wiles, T. J., 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=Modernized+tools+for+streamlined+genetic+manipulation+and+comparative+study+of+wild+and+diverse+proteobacterial+lineages.">Modernized tools for streamlined genetic manipulation and comparative study of wild and diverse proteobacterial lineages.</a> mBio. 9 (5), 01877 (2018).</li><li>Ronda, C., Chen, S. P., Cabral, V., Yaung, S. J., Wang, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomic+engineering+of+the+mammalian+gut+microbiome+in+situ.">Metagenomic engineering of the mammalian gut microbiome in situ.</a> Nature Methods. 16 (2), 167-170 (2019).</li><li>Leonard, S. P., 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=Genetic+engineering+of+bee+gut+microbiome+bacteria+with+a+toolkit+for+modular+assembly+of+broad-host-range+plasmids.">Genetic engineering of bee gut microbiome bacteria with a toolkit for modular assembly of broad-host-range plasmids.</a> ACS Synthetic Biology. 7 (5), 1279-1290 (2018).</li><li>Kutscher, L. M., Shaham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+and+reverse+mutagenesis+in+C.+elegans.">Forward and reverse mutagenesis in C. elegans.</a> WormBook: The Online Review of C. elegans Biology. , 1-26 (2014).</li><li>Mok, C. A., 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=MIP-MAP:+High-Throughput+mapping+of+Caenorhabditis+elegans+temperature-sensitive+mutants+via+molecular+inversion+probes.">MIP-MAP: High-Throughput mapping of Caenorhabditis elegans temperature-sensitive mutants via molecular inversion probes.</a> Genetics. 207 (2), 447-463 (2017).</li></ol>

在多项研究中，流式蚯蚓法已被用于表征秀丽隐杆线虫基因和抗病原体定植和毒性的途径21,22。在这里，提出了一种高通量的方法，该方法使用秀丽隐杆线虫来研究肠道微生物组如何调节其宿主生理学。与使用集落形成单位 （CFU） 或 16S rRNA 扩增子测序9、16、17、<sup clas...

动物的进化受到微生物的持续影响1.从环境中的各种微生物中，动物宿主获得了特定的伙伴2 ，这些伙伴扩展了宿主的能力并驱动其生理学和对疾病的易感性3。例如，肠道微生物组的宏基因组分析揭示了富含代谢类别的微生物基因，这些基因可能在肥胖小鼠中赋予更大的能量收集和储存4，其中许多也存在于人类肠道微生物组5中。尽管微生物组的复杂性和宿主系统对大规模筛选的可处理性阻碍了进展，但仍然非常需要建立因果关系并确定微生物组影响的分子决定因素。
模式生物秀丽隐杆线虫提供了一个平台，以促进对微生物组和宿主生理学之间联系的分子理解。秀丽隐杆线虫拥有 20 个具有粘膜层和绒毛结构的肠细胞。这些细胞配备了丰富的化学感受器基因，这些基因可以感知微生物产物并产生可能调节其肠道定植者的抗菌分子6,7。秀丽隐杆线虫的这种保守生物学导致了大量调节肠道微生物的宿主信号传导的发现，包括胰岛素信号传导、TGF-β 和 MAP 激酶 8,9,10。
秀丽隐杆线虫在发育过程中利用微生物作为其生长的饮食，并利用成虫的微生物组。随着年龄的增长，一些微生物可能会在肠腔中过度积累，宿主与微生物的关系从共生转变为发病机制11。在它们的自然栖息地中，秀丽隐杆线虫会遇到各种各样的细菌物种12,13。对在自然栖息地（腐烂的水果、植物茎和动物载体）收集的代表性样本进行16S rDNA测序，发现秀丽隐杆线虫的天然微生物组以4个细菌门为主：变形菌门、拟杆菌门、厚壁菌门和放线菌门。 在这些分区中，基于栖息地的细菌多样性和丰富度存在很大差异12,13,14,15。已经建立了几个明确的群落，包括 63 个成员 （BIGbiome）16 和 12 个成员 （CeMbio） 集合，代表了为秀丽隐杆线虫研究社区创建的顶级微生物组属17。微生物组和成分菌株都可以对秀丽隐杆线虫的生理机能产生不同的影响，例如体型、生长速度和应激反应 9,16,17。这些研究为将秀丽隐杆线虫确立为微生物组研究的模型提供了资源和示例。
这里介绍了一个基于大型颗粒分选机（LPS）的工作流程（图1），该工作流程利用 秀丽隐杆线 虫系统在群体规模上同时测量微生物组组成和宿主生理学的基本测量。从微生物方面来看，该工作流程适用于组装定义的微生物组或单个微生物，以测试群落的稳健性和可塑性，并增加微生物相互作用。从宿主侧来看，该工作流程可实现高通量检测，以测量微生物组中荧光微生物的定植水平，并在发育、体型和繁殖方面获得宿主生理读数。综上所述， 秀丽隐杆 线虫微生物组模型能够进行高通量筛选，以确定调节宿主生理学的代谢和遗传决定因素。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL conical bottom centrifuge tubes</td>
			<td>VWR</td>
			<td>10026-076</td>
			<td></td>
		</tr>
		<tr>
			<td>96 deep-well plates (1 mL)</td>
			<td>Axygen</td>
			<td>P-DW-11-C</td>
			<td></td>
		</tr>
		<tr>
			<td>96 deep-well plates (2 mL)</td>
			<td>Axygen</td>
			<td>P-DW-20-C</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Costar plate</td>
			<td>Corning</td>
			<td>3694</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Millipore Sigma</td>
			<td></td>
			<td>Standard bacteriology agar is also sufficient.</td>
		</tr>
		<tr>
			<td>Aspirating manifold</td>
			<td>V&#38;P scientific</td>
			<td>VP1171A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Clorox</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach solution&#160;</td>
			<td></td>
			<td></td>
			<td>Mix Bleach with 5M Sodium hypochlorite 2:1 (v/v)</td>
		</tr>
		<tr>
			<td>Cell Imaging Multimode Reader</td>
			<td>Biotek</td>
			<td>Cytation 5</td>
			<td>Bacterial OD measurement</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo scientific&#160;</td>
			<td>Sorvall Legend XTR</td>
			<td>For 96 well plate and conical tubes</td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>Nikon</td>
			<td>TiE</td>
			<td></td>
		</tr>
		<tr>
			<td>ggplot: Various R Programming Tools for Plotting Data.</td>
			<td>R package</td>
			<td>Version 3.3.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Particle Autosampler</td>
			<td>Union Biometrica</td>
			<td>LP Sampler</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Particle Sorter</td>
			<td>Union Biometrica</td>
			<td>COPAS Biosorter</td>
			<td></td>
		</tr>
		<tr>
			<td>Levamisole</td>
			<td>Fisher</td>
			<td>AC187870100</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysogeny Broth (LB)</td>
			<td>RPI</td>
			<td>L24066</td>
			<td>Standard LB home-made recipes using Bacto-tryptone, yeast extract, and NaCl are also sufficient.</td>
		</tr>
		<tr>
			<td>M9 solution&#160;</td>
			<td></td>
			<td></td>
			<td>22 mM KH2PO4 monobasic, 42.3 mM Na2HPO4, 85.6 mM NaCl, 1 mM MgSO4</td>
		</tr>
		<tr>
			<td>Nematode Growth Medium</td>
			<td>RPI</td>
			<td>N81800-1000.0</td>
			<td>1 mM CaCl2, 25 mM KPO4 pH 6.0, 1 mM MgSO4 added after autoclaving.</td>
		</tr>
		<tr>
			<td>RStudio</td>
			<td>GNU</td>
			<td>Version 1.3.1093</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hypochlorite</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>5M NaOH</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 10 cm diameter petri dishes</td>
			<td>Corning</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 12-well plates</td>
			<td>VWR</td>
			<td>10062-894</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 24-well plates</td>
			<td>VWR</td>
			<td>10062-896</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 6 cm diameter petri dishes</td>
			<td>Corning</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
	</tbody>
</table>

application of flow vermimetry for quantification and analysis of the caenorhabditis elegans gut microbiome

肠道微生物组的组成会对动物的整个发育和生命过程中的宿主生理机能产生巨大影响。测量微生物组的组成变化对于确定这些生理变化之间的功能关系至关重要。 秀丽隐杆线虫 已成为一种强大的宿主系统，用于研究宿主-微生物组相互作用的分子驱动因素。凭借其透明的身体计划和荧光标记的天然微生物，可以使用大型颗粒分选机轻松量化单个 秀丽隐杆线虫 动物肠道微生物组内微生物的相对水平。在这里，我们描述了微生物组的实验设置、所需生命阶段线 虫 种群的收集和分析、分选机的操作和维护以及所得数据集的统计分析的程序。我们还讨论了基于目标微生物优化分选机设置的注意事项，为 秀丽隐杆 线虫生命阶段开发有效的门控策略，以及如何利用分选机功能根据肠道微生物组组成丰富动物种群。潜在应用的示例将作为协议的一部分进行介绍，包括扩展到高通量应用的潜力。

定义成虫和幼虫种群门 在这里，同步的秀丽隐杆线虫 L1 生长在接种有大肠杆菌 OP50 （Eco） 的 NGM 平板上，这是一种标准的实验室日粮。在20°C下生长96小时或120小时后收集秀丽隐杆线虫种群进行LPS分析（图2A）。灭绝点图（EXT，身体密度的代理）与飞行时间（TOF，体长的代理）创建了两个视觉上分离的动物云。每个点代表一只动物，与幼虫...

Watch this Scientific Journal Video about Application of Flow Vermimetry for Quantification and Analysis of the Caenorhabditis elegans Gut Microbiome at JoVE.com

Application of Flow Vermimetry for Quantification and Analysis of the Caenorhabditis elegans Gut Microbiome

秀丽隐杆线虫 是一种强大的模型，用于研究驱动宿主-微生物组相互作用的分子决定因素。我们提出了一个高通量管道，分析肠道微生物组定植的单一动物水平以及 秀丽隐杆 线虫生理学的关键方面。

流动蚯蚓法在 秀丽隐 杆线虫肠道微生物组定量分析中的应用

The composition of the gut microbiome can have a dramatic impact on host physiology throughout the development and the life of the animal. Measuring ...

肠道微生物组在塑造宿主的生理机能方面起着重要作用。特别是，该协议允许大规模评估单个细胞动物中的荧光肠道微生物定植水平。这种方法的一个主要优点是能够实时监测活体动物的肠道微生物组，从而可以识别任何变化并将其与宿主的生理机能相关联。演示该程序的将是我实验室的研究助理达娜·布莱克本（Dana Blackburn）。首先用一到两毫升的M9-TX将蠕虫从细菌草坪上洗掉。将蠕虫转移到灭菌的两毫升96孔深板中。将它们以 300 G 沉淀一分钟，然后使用吸气歧管除去液体。使用1.2毫升多通道移液器，通过移液几次混合并以300G离心1分钟，将1.8毫升M9-TX加入深孔板的每个孔中。最终洗涤后，使用吸气歧管将每个孔中的体积增加到 100 微升。向每个孔中加入 100 微升 10 毫摩尔左旋咪唑和 M9-T，让蠕虫麻痹 5 分钟。然后向每个孔中加入 4% 漂白剂溶液两分钟，以减少细菌团块。漂白剂处理后，将M9-TX加入板的每个孔中以洗涤蠕虫。第二次洗涤后离心板并吸出至最终体积为100微升。将蠕虫转移到平底96孔板上，该孔板含有150微升10毫摩尔左旋咪唑和M9-TX。将蠕虫密度保持在每孔 50 到 100 条蠕虫，如果种群过于拥挤，则将蠕虫分成多个孔。打开空压机、电脑和LPS仪器。检查并清空废液箱。如果体积低，请检查并重新填充护套和水箱。确保 250 微米流体和光学核心组件就位。连接并打开自动进样器。打开自动进样器仪器软件，打开自动进样器和LPS软件。在LPS软件窗口中，转到“文件”并单击“新建实验”，然后再次选择“文件”并单击“新建样品检查”。启用激光器以打开 488 和 561 纳米激光器（如果尚未完成）。现在，使用 LPS 软件窗口中的飞行时间、消光和荧光通道创建采集点图模板。在图形主体中单击鼠标右键以修改缩放比例。接下来，加载对照样本。在自动进样器窗口中，选择 prime，转到文件，单击打开脚本以选择正确的内置脚本，然后单击确定。转到取样器软件菜单上的板模板，选择所需的孔进行分析。将板加载并固定到自动进样器载物台上后，按自动进样器窗口上的运行板并在出现提示时保存文件，然后单击 LPS 软件窗口中顶部功能区上的存储当前数据按钮并再次保存数据。打开 LPS 软件。导航到“文件”并选择“新建实验”，然后返回“文件”并单击“新建样本”。创建一个点图，其中飞行时间在 X 轴上，消光时间在 Y 轴上。创建另一个点图，其中飞行时间在 X 轴上，红色在 Y 轴上。确保检查启用激光器以激活 488 和 561 纳米激光器。将控制样品管放在样品端口上，然后在采集和分配对话框中单击采集。出现提示时，请确保保存文件。一旦测量了足够多的蠕虫来区分种群，在显示飞行与灭绝时间的点图中选择中止。在代表成年人口的区域周围画一个门。然后导航到视图，单击“门控层次结构”，并检查荧光门是否正确列在“成年人群门”下。在飞行时间与红点图中，在高红值和低红值区域周围创建门，以突出显示显示成虫中微生物组定植的感兴趣区域。优化设置后，继续文件并单击保存实验，然后单击文件并选择保存样品。要加载收集设备，请选择加载板 A，以允许收集阶段移动到加载收集管或 96 孔板的准备位置。要分配到 96 孔板，请转到设置部分并选择板，然后单击校准板并选择 96 孔板。接下来，选择要对蠕虫进行分类的孔，并输入要分类到每个孔中的对象数量。请务必同时指定感兴趣的门控区域。如果有多个浇口区域被分类到同一个板中，请确保选中每个孔标有浇口的框。单击“确定”以保存更改。对编号进行排序并分配位置后，将样品加载到样品端口上，然后单击采集和分配对话框中的填充板按钮，开始分配到 96 孔板中。与幼虫相比，在成虫中观察到更高的延伸和飞行时间值。两日龄成虫的后代以L1和L2期为主，而三日龄成虫的大多数后代达到L3和L4期。当在大肠杆菌上生长时，与第二天相比，第三天的飞行时间和延伸值增加。当在赭石、BH 3 和混合培养物上生长时，Cenor abdidas allegands 在飞行时间和延伸值方面表现出差异。在混合条件下观察到赭色 BH3 定植增加，而不是单独在赭色 BH3 中。相比之下，绿色荧光值表明混合条件下的OP50定植程度低于单独使用OP50。蠕虫严重偏向 Y 轴，表明大多数蠕虫的 OP50 定植率较低，而 ochrobactrum、BH3 定植水平在种群中均匀分布。还观察到赭齿形、BH3定植水平与宿主发育（如体密度）和繁殖模式差异之间的关系。在两个成员混合物上生长的三天龄成虫，微生物组表现出广泛的RFP强度，表明组内赭形菌定植的个体差异。显示了包含来自高低 RFP 闸门的 15 个分类和单个蠕虫的孔的 RFP 图像。最重要的是记住对所有步骤使用适当的控件，以确保协议和机器正常工作。实时，具有特定特征的动物可用于从宿主或微生物突变体库中分离菌株，以鉴定调节微生物后相互作用的基因。分离的动物也可用于单动物或富集的基于表型库的下游分析，如RNA-seq等。真的，机会真的是无穷无尽的。

Research

JoVE Journal

Biology

Application of Flow Vermimetry for Quantification and Analysis of the Caenorhabditis elegans Gut Microbiome

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

测量线虫寿命在96孔板

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

科研

生物学

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

前列腺素的提取与分析<em&gt;线虫</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

栽培<em&gt;秀丽隐杆线虫</em&gt;在三个维度实验室

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

尼罗红与油红 O 染色法测定秀丽线虫的脂质丰度及脂质分布评价

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

卡诺尔哈布炎细胞外囊泡的纯化与分析

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

使用 卡诺哈布迪炎埃莱甘斯 筛选组织特异性查佩龙相互作用

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Automating Aggregate Quantification in Caenorhabditis elegans

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

秀丽隐杆线虫种系内突触复合体的超分辨率显微镜

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures ...

流动蚯蚓法在 秀丽隐 杆线虫肠道微生物组定量分析中的应用

流动蚯蚓法在秀丽隐杆线虫肠道微生物组定量分析中的应用