Name: plate-based large-scale cultivation of caenorhabditis elegans: sample preparation for the study of metabolic alterations in diabetes
Uploaded: 2018-08-24T14:00:00.000+00:00
Duration: 5 min 8 s
Description: Watch this Scientific Journal Video about Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes at JoVE.com

这项研究得到德意志 Forschungsgemeinschaft (DFG) 在 IRTG 1874 "糖尿病微血管并发症" 和 CRC 1118 "反应性代谢物作为糖尿病晚期并发症的原因" 的支持。N2 和 CL2166 的线虫菌株由 CGC 提供, 由 NIH 研究基础设施项目办公室 (P40 OD010440) 资助。

注: 本协议分为五节。在1–3节中, 提出了大规模养殖C 型线虫的主要协议。4和5节为评估糖尿病代谢产物中出现的代谢产物提供了附加的协议。详细来说, 第1节描述了一个普通的大范围的板块文化。2节重点介绍了大量的C 线虫的转移, 而3节则解释了大规模样品的收获。4节解释了从样品中分离出的蛋白质, 5 节描述了随后的液相色谱-串联质谱 (LC-ms) 分析的样品制备方法。
1. 板材上的大范围文化
<ol><li>一般情况下, 工作在无菌罩下或在明火旁边以避免污染。</li>
	<li>
要培养蠕虫直到它们到达年轻的成人阶段, 请按照以前发布的7号协议进行以下修改。
	<ol>
<li>使用活大肠杆菌 OP50 细菌喂养线虫, 并准备400µM fluorodeoxyuridine 琼脂 (不氨苄西林/FUdR) 进行实验。</li>
		<li>为同步人群的准备, 使用非浓缩 OP50, 并为成功的实验与成人, 使用3.3x 浓缩 OP50 通过去除70% 的上清。</li>
		<li>建议每日喂食间隔。在评估氧化应激时, 不同的进食间隔和容积可能会影响结果。</li>
		<li>为接种, 采取一个板材与几个线虫并且切开它成片断大约 0.5 x 1 cm2与手术刀。将两到三块转移到线虫生长介质 (NGM) 板上, 直径为60毫米, 含非浓缩 OP50。</li>
		<li>在适宜于应变的温度下孵化板材 (野生型 N2 应在20摄氏度培养), 直到鸡蛋在盘子上可见。</li>
		<li>用2毫升的 M9 缓冲液冲洗掉大部分的卵和成年动物, 并在15毫升管中收集它们。离心机悬浮在 800 x g为2分钟。同时, 开始准备漂白液。</li>
		<li>取出离心管, 用10毫升吸管取出 M9 缓冲器。添加10毫升的漂白溶液和混合在漩涡, 直到成人线虫裂解。这通常采取大约 5–7 min, 但不应该花费超过15分钟或蛋不会完全地以后开发在实验中。</li>
		<li>离心机悬浮在 800 x g为2分钟, 洗3次与10毫升 M9 缓冲, 以清除鸡蛋从漂白溶液。</li>
		<li>现在添加150µL 的蛋悬浮在 OP50 板上。每个盘子上都要有200–300xg 蛋的密度。等到线虫到达成年阶段。 注: 所述实验所用的 M9 缓冲器 (pH 7.0) 由以下物质组成:22 毫米,2PO4, 42 毫米 Na2HPO4, 和86毫米氯化钠。热处理后加入1毫米 MgSO4。漂白溶液含有0.5 米氢氧化钠, 2.3 毫米 NaOCl 在蒸馏水中解决。</li>
		<li>准备无菌 M9 缓冲剂, 不育的吸管提示用切尖冲洗蠕虫, 和15毫升管收集他们在。</li>
		<li>在盘子上涂上大约1毫升的 M9 缓冲器, 轻轻地将吸管的线虫放在盘子上, 轻柔地散发缓冲器。</li>
		<li>现在, 收集管中的线虫。直接应用150µL 悬浮在板材 (种子与 OP50) 与切开的吸管提示, 并且显微镜下评估线虫的密度 (推荐: 50–100线虫每60毫米板材)。</li>
		<li>如果它们过于致密, 稀释悬浮与 M9 缓冲, 并评估它在显微镜下, 直到达到预期的密度。如果产量太少, 让蠕虫安定下来或离心机悬浮十年代在 800 x g去除液体。 注: 一个充分的密度的例子在图 1A (宏观) 和1B (微观) 在20X 增量给。按照所描述的经验进行。</li>
		<li>请记住, 蠕虫很快就会安定下来。为了保证板材之间的均匀分布, 在每叠板 (第4-5 板) 后反转管。 注: 切割吸管尖端, 以及温和摆动和洗涤的盘子, 减少剪应力。</li>
	</ol>
</li>
</ol>2.大量秀丽线虫的转移
<ol><li>用大约500µL 的 M9 缓冲剂洗涤线虫, 这取决于盘子的年龄和干燥程度。使用相同的缓冲, 重复分离线虫, 直到大多数动物被收集在悬浮。</li>
	<li>慢慢地用割下的吸管把线虫带上, 并将它们转移到 OP50 准备好的盘子上。让盘子干。 注意: 注意不要比推荐的卷应用得多。特别是在长期的实验中, 板块可能无法耐受, 琼脂也会断裂, 使线虫爬入裂缝中。</li>
</ol>3. 大秀丽线虫样品的收获
<ol><li>根据所选的方法, 开始实验, 有足够数量的C. 线虫。对于 LC-质谱/ms 分析, 每组20板 (60 毫米 x 15 毫米), 每片约100个线虫, 以确保每样蛋白质至少有200µg 的产量。 注: 本议定书的这一部分不再需要在无菌条件下工作, 因为线虫将被收集和冻结。</li>
	<li>在样品收集的当天, 准备液氮以对收集的样品进行冻结。在冰上保持无菌 M9 缓冲, 以减缓线虫的新陈代谢。</li>
	<li>用1毫升的 M9 缓冲器, 轻轻地旋转盘子, 从盘子里洗去线虫。这避免收集已故的线虫和多数蛋, 如果有任何礼物, 因为他们坚持细菌和琼脂。</li>
	<li>把音量调到15毫升管上。</li>
	<li>收集完样品后, 等待线虫在 800 x g处简单地沉降或离心管, 并取出大部分 M9 缓冲器。用大约10毫升的 M9 (10x 样本量) 冲洗样品3x 以除去任何细菌。 注: 为确保所有已死亡的线虫被除去, 蔗糖浮选是另一种选择。然而, 这是不适合的显示实验15。蔗糖是水解葡萄糖和果糖。在所描述的实验中, 评价葡萄糖的作用, 以促进慢性糖尿病的生化变化。因此, 蔗糖浮选可能会混淆结果。</li>
	<li>准备一个1.5 毫升管与1毫升的 M9 缓冲器和一个空1.5 毫升管每样。将球团与玻璃吸管从15毫升管转移到空1.5 毫升管。这一步骤对于确保定量转移至关重要。 注意: 不像以前的步骤, 这里的线虫更集中。由于可能坚持塑料吸管的提示, 损失的大部分样品是预期的。因此, 用玻璃吸管代替。</li>
	<li>转移后, 使用玻璃吸管从第二个1.5 毫升管中拿起1毫升的 M9 缓冲器, 从吸管壁上冲洗线虫。另外, 从15毫升管中清洗剩余的线虫, 并将其转移到样品管。</li>
	<li>离心样品管在 1.9万 x g 1 分钟, 然后删除尽可能多的上清。</li>
	<li>用 Parafilm 封住管子, 并立即将其冻结在液氮中。贮存管在-80 摄氏度, 直到裂解制剂。</li>
</ol>4. 质谱分离蛋白
注意: 这里描述的蛋白质分离也可以用于其他化验 (例如, 西方印迹)。
<ol><li>对冷冻样品, 添加相同数量的0.5 毫米氧化锆珠和至少2x 的裂解液容量含有蛋白酶抑制剂鸡尾酒。 注: 用100µL 缓冲剂进行了描述的线虫-蛋白质相关分析。裂解200µL 的缓冲液。</li>
	<li>在速度9上启动1分钟的均质机。确保样品完全裂解。如果没有完全裂解, 请重复此步骤。</li>
	<li>离心样品30分钟在4°c 在 1.9万 x g。将上清液转移到冰上的新管上, 将其储存在-80 摄氏度, 直到进一步测量。 注: 所述实验所用的非变性缓冲器由以下物质组成:20 毫米三盐酸 (pH 8)、137毫米氯化钠、1% 海卫 x 和2毫米 EDTA。 注: 遵循本议定书的步骤, 并应用了建议的规模, 蛋白质颗粒从大约1000线虫的产量应约500µg。有关更多比较, 请参阅图 2和代表结果。</li>
</ol>5. 液相色谱-串联质谱测量样品的制备
注: 根据感兴趣的参数, 样品准备会有所不同。本议定书的重点是丙酮醛和年龄确定。
<ol><li>
用 12-diaminobenzene (DB) 的衍生化方法测量胞内丙酮醛, 如前所述9。
	<ol>
<li>融汇样品与20µL 冰冷 20% (重量/卷) 乙酸酸在 0.9% (重量/卷) 氯化钠和80µL 水在1.5 毫升管子。</li>
		<li>用内部标准 (13C3毫克; 400 nM) 将5µL 的样品钉在一起, 然后用涡流将它们混合, 然后在 2.1万 x g处将其离心为5摄氏度。</li>
		<li>将上清的35µL 转移到含有200µL 玻璃插入物的 HPLC 样品瓶中。</li>
		<li>将5µL 3% 叠氮化钠 (重量/卷) 添加到每个样品, 后跟10µL 0.5 毫米 DB 在200毫米 HCl 中含有0.5 毫米 diethylenetriaminepentaacetic 酸 (DETAPAC) 在水中。</li>
		<li>在室温下孵化4小时的样品, 免受光照。</li>
		<li>如前文所述, 分析样品的 LC-ms/毫秒,9。 注: 对于年龄的测量, 请将该协议第4节所述的蛋白质分离出来。</li>
	</ol>
</li>
	<li>
用布拉德福德10测定 (解冻) 裂解物的蛋白质浓度。
	<ol>
<li>在磷酸盐缓冲盐水 (PBS) 中, 用牛血清白蛋白 (BSA) 解决的标准曲线整除数30µL 的含量: 0;0.1;0.2;0.3;0.4;0.6;0.8;和1.0 毫克/dL。 注: 如果样品是按照建议的尺寸制备的 (见步骤 4.1, 用于制备具有200µL 缓冲液的裂解剂), 估计浓度应在大约2–7毫克/毫升范围内。在这种情况下, 在测量之前用 PBS 稀释样品1:10。 注意: 对于第一个使用此方法的实验, 建议对不同稀释 (1、1:10 和 1:100) 中的样品进行测量。由于没有确切的确定线虫数量, 不同的研究人员的产量可能有所不同。</li>
		<li>使用透明的96井板 (聚苯乙烯) 和吸管10µL 的每个标准浓度和样品在选定的稀释中重复的水井。</li>
		<li>用蒸馏水 (蒽醌) 稀释蛋白质染料试剂浓缩1:5。工作台)并添加200µL 的稀释到每个样品上的盘子。在室温下孵育10分钟的盘子。</li>
		<li>测量30分钟内的吸光度在595毫微米与一个板块阅读器。样品可以从计算的标准曲线中插值。该方法的详细资料在文献10中得到了广泛的描述。</li>
	</ol>
</li>
	<li>
测量细胞内的年龄, 如前所述11,12。
	<ol>
<li>在 10 kDa 离心过滤装置中, 用离心清洗总蛋白提取物 (约200µg) 3x。</li>
		<li>加入10µL 100 毫米盐酸, 10 µL 胃蛋白酶 (2 毫克/毫升在20毫米 hcl) 和10µL 麝香 (2 毫克/毫升在20毫米 hcl) 到恢复的蛋白质 (大约100µL) 和孵化样品在37°c 为 24 h。</li>
		<li>通过添加12.5 µL 0.5 米磷酸钾缓冲液 (ph 7.4) 和5µL 260 毫米的 KOH, 中和并缓冲样品在 pH 值7.4。</li>
		<li>添加10µL 链霉蛋白酶 E [2 毫克/毫升10毫米磷酸钾缓冲液 (pH 7.4)] 和10µL 青霉素-链霉素溶液, 并孵化样品在37°c 24 h。</li>
		<li>添加10µL 的氨基 [2 毫克/毫升10毫米磷酸钾缓冲 (ph 7.4)] 和10µL 脯肽酶 [2 毫克/毫升在10毫米磷酸钾缓冲 (pH 7.4)], 并孵化样品在37°c 48 h。</li>
		<li>分析所产生的水解物的 LC-ms/毫秒, 如前所述12。</li>
	</ol>
</li>
</ol>

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(64), e4019 (2012).</li><li>Lagido, C., McLaggan, D., Glover, L. 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+Screenable+In+Vivo+Assay+for+Mitochondrial+Modulators+Using+Transgenic+Bioluminescent+Caenorhabditis+elegans.">A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans.</a> Journal of Visualized Experiments. (105), e53083 (2015).</li><li>Takamiya, S., Mita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+purification+of+active+liquid-cultured+Caenorhabditis+elegans+using+a+modified+Baermann+apparatus.">Large-scale purification of active liquid-cultured Caenorhabditis elegans using a modified Baermann apparatus.</a> Parasitology International. 65 (5 Pt B), 580-583 (2016).</li><li>Cornaglia, M., Lehnert, T., Gijs, M. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+systems+for+high-throughput+and+high-content+screening+using+the+nematode+Caenorhabditis+elegans.">Microfluidic systems for high-throughput and high-content screening using the nematode Caenorhabditis elegans.</a> Lab Chip. 17 (22), 3736-3759 (2017).</li><li>Zhu, G., Yin, F., Wang, L., Wei, W., Jiang, L., Qin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+type+2+diabetes-like+hyperglycemia+in+C.+elegans+on+a+microdevice.">Modeling type 2 diabetes-like hyperglycemia in C. elegans on a microdevice.</a> Integrative Biology. 8 (1), 30-38 (2016).</li></ol>

作者没有什么可透露的。

该协议为大型线虫的大规模培养提供了可靠的途径, 获得了定量的结果。文献的结果可以复制, 如代表性结果所示。尽管这个用于收集大型线虫样本的协议似乎是一种直截了当的方法, 但还是有一定的缺陷需要考虑。关于线虫种群的同步问题, 本议定书描述了一种方法, 即用次氯酸钠和氢氧化钠漂白种群, 以破坏线虫, 并完全收获卵子。必须考虑到这种方法可能不适合所有实...

蛋白质分析, 如西方的印迹或质谱, 需要蛋白质的毫克。这种产量需要大规模培养数以百计的线虫, 这可以通过液体培养或固体介质通过洗涤转移线虫。流体和剪切应力诱导上皮钠通道 (ENaC) 的表达, 通过增加钠的吸收增加渗透应力, 可能改变线虫的寿命, 影响代谢分析1.因此, 该协议中的一些关键步骤, 以板块为基础的方法采取减少应力影响实验的变异性考虑。另一方面, 液体培养影响了线虫的表型, 复杂了2线虫的培养和准确数量的收集。此外, 活性物质可以改变的媒介成分, 并可能分布不均匀, 然后到达线虫。关于液体培养的局限性, 本议定书提供了一种新的方法来培养大规模的线虫样本。
线虫是一个模型有机体, 具有独特的网络302神经细胞, 占其所有细胞的 1/33。自引入科学以来, 许多同源和同源基因被描述, 放大它作为医学研究模型的价值。最近, 糖尿病视网膜病变 (前血管损伤) 的神经功能损害的证据已经提出4。线虫缺乏血管, 但包含一个独特的神经网络, 这使得它成为一个合适的模型来调查神经元的变化, 除了血管的。因此, 对于研究糖尿病并发症的一般机制,线虫有特殊的兴趣。糖尿病并发症的生化变化涉及年龄的形成, 这进一步影响了 ROS 的形成, 以应对高血糖5。年龄被发现在C. 线虫和贡献神经损伤6。慢性疾病通常是由复杂的多基因过程引起的, 需要多参数方法来评估其基本机制, 这里举例说明糖尿病并发症的评估。该协议可用于同时获取多个参数, 以及随后使用。通过省略液体与固体培养基之间的形态和代谢差异, 可以提高多参数方法的可比性和重现度。

<table><tbody><tr><td>&lt;em&gt;E. coli &lt;/em&gt;OP50</td><td>CGC</td><td>n/a</td><td></td></tr><tr><td>&lt;em&gt;C. elegans &lt;/em&gt;N2</td><td>CGC</td><td>n/a</td><td></td></tr><tr><td>&lt;em&gt;C. elegans &lt;/em&gt;CL2166</td><td>CGC</td><td>n/a</td><td></td></tr><tr><td>Petri dish, 60 mm x 15 mm</td><td>Greiner One</td><td>628161</td><td></td></tr><tr><td>Volumetric pipet, glas, 10 mL</td><td>Neolab</td><td>E-0413</td><td></td></tr><tr><td>Proteinase inhibitor cocktail tablets</td><td>Roche</td><td>04693124001</td><td></td></tr><tr><td>&lt;u&gt;Non-denaturing lysate buffer:&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Tris-HCl, pH 8</td><td>Sigma</td><td>T3253</td><td></td></tr><tr><td>Sodiumchloride (NaCl)</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X-100</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma</td><td>E5391</td><td></td></tr><tr><td>96-well plates, transparent bottom</td><td>Brand</td><td>781611</td><td></td></tr><tr><td>Infinite M200, plate reader</td><td>Tecan</td><td>30017581</td><td></td></tr><tr><td>Zirconium Oxide Beads, 0.5 mm</td><td>Next advance</td><td>ZROB05-RNA</td><td></td></tr><tr><td>Bullet Blender, homogenizer</td><td>Next advance</td><td>BBX24</td><td></td></tr><tr><td>Pepsin from porcine gastric mucosa</td><td>Sigma</td><td>P6887</td><td></td></tr><tr><td>Thymol</td><td>Sigma</td><td>T0501</td><td></td></tr><tr><td>Pronase E/ Protease from &lt;em&gt;Streptomyces griseus &lt;/em&gt;</td><td>Sigma</td><td>P6911</td><td></td></tr><tr><td>Penicillin-Streptomycin solution</td><td>Sigma</td><td>P43339</td><td></td></tr><tr><td>Prolidase from Porcine Kidney</td><td>Sigma</td><td>P6675</td><td></td></tr><tr><td>Aminopeptidase from&lt;em&gt; Aeromonas proteolytica&lt;/em&gt;</td><td>Sigma</td><td>A8200</td><td></td></tr><tr><td>Amicon Ultra-0.5 Centrifugal Filter Unit</td><td>Merckmillipore</td><td>UFC501096</td><td></td></tr><tr><td>Basic Materials for plate culture are described in Reference &lt;sup class=&quot;xref&quot;&gt;6&lt;/sup&gt;.</td><td></td><td></td><td></td></tr></tbody></table>

plate-based large-scale cultivation of caenorhabditis elegans: sample preparation for the study of metabolic alterations in diabetes

在琼脂盘子上以大规模的方式培养秀丽线虫(线虫) 是费时费力的。该协议描述了一个简单而廉价的方法, 以获得大量的动物, 以分离蛋白质, 进行了西方印迹, 质谱, 或进一步蛋白质组学分析。此外, 在相同的培养条件下, immunostainings 线虫数量的增加和多种分析的整合也很容易实现。另外, 在不同实验条件下的板材之间的转移是方便的。在板块文化中常见的技术是用一根铂丝和用手术刀转移填充的琼脂块来转移单一的线虫.然而, 随着线虫数量的增加, 这些技术变得过于耗时。该协议描述了大规模的C 线虫文化, 包括许多步骤, 以尽量减少样本准备对蠕虫的生理影响。液体和剪切应力可以改变线虫的寿命和代谢过程, 因此需要详细描述关键步骤, 以检索可靠和可重现的结果。线虫是一种模型有机体, 由神经细胞组成, 可达 1/3, 但缺乏血管, 从而提供了单独研究独立于血管控制的神经元改变的可能性。最近, 在血管改变之前发现早期变性糖尿病视网膜病变。因此, 对于研究糖尿病并发症的一般机制,线虫有特殊的兴趣。例如, 在重现性中发现了晚期糖基化终产物 (年龄) 和活性氧 (ROS) 的增加形成. 通过对糖尿病引起的生化变化的研究, 本文介绍了处理足够大小样本以进行更广泛调查的协议。一般而言, 此协议对于需要大的C 线虫数和液体培养不合适的研究是有用的。

这里给出了创建大规模的C. 线虫文化在糖尿病研究中应用的例子。将参数与单个动物进行关联, 而不是将其规范化为总的蛋白质浓度是很有意义的。在需要少量线虫的化验中, 这可以很容易地通过计数线虫来完成。对于一个大规模的C. 线虫文化, 涉及数以百计的线虫每实验组, 这种方法是不方便的。在图 2中, 显示了线虫数量与蛋白?...

Watch this Scientific Journal Video about Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes at JoVE.com

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

本协议描述了一种在固体培养基上大规模培养秀丽线虫的方法。作为液体培养的替代品, 该协议允许在板基培养条件下获得不同尺度的参数。这增加了结果的可比性通过省略液体和固体媒介文化之间的形态学和新陈代谢的区别。

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

plate-based-large-scale-cultivation-caenorhabditis-elegans-sample

Research

JoVE Journal

Biochemistry

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

6.3K 次观看.  Heidelberg University. 本协议描述了一种在固体培养基上大规模培养秀丽线虫的方法。作为液体培养的替代品, 该协议允许在板基培养条件下获得不同尺度的参数。这增加了结果的可比性通过省略液体和固体媒介文化之间的形态学和新陈代谢的区别。

视频: Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

大规模混合阶段 卡诺哈布迪蒂斯电子 人种群的文化和分析

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...

科研

遗传学

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

癌症研究

A Caenorhabditis elegans Model System for Amylopathy Study

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

一<em&gt;线虫</em&gt;研究Amylopathy模型系统

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

神经科学

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

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

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

We describe protocols for using the wide field-of-view nematode tracking platform (WF-NTP), which enables high-throughput phenotypic characterization of large populations of Caenorhabditis elegans. These protocols can be used to characterize subtle behavioral changes in mutant strains or in response to pharmacological treatment in a highly scalable fashion.

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 ...

免疫与感染

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

培养<em&gt;秀丽隐杆线虫</em &gt;在无菌液体介质与创造转基因蠕虫通过微粒轰击

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

生物学

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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 know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

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 ...

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.

微生物分离株的高通量筛选对 秀丽隐杆线虫 健康的影响

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on ...

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

在固体培养基上长期培养单个 秀丽隐杆线虫 ，用于纵向荧光监测和厌恶干预

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, rapid reproduction cycle (~3 days), short lifespan (~3 weeks), and numerous available tools for genetic manipulation and molecular analysis. The most common approach for conducting aging studies in C. elegans, including survival analysis, involves culturing populations of tens to hundreds of animals together on solid nematode growth media (NGM) in Petri plates. While this approach gathers data on a population of animals, most protocols do not track individual animals over time. Presented here is an optimized protocol for the long-term culturing of individual animals on microfabricated polydimethylsiloxane (PDMS) devices called WorMotels. Each device allows up to 240 animals to be cultured in small wells containing NGM, with each well isolated by a copper sulfate-containing moat that prevents the animals from fleeing. Building on the original WorMotel description, this paper provides a detailed protocol for molding, preparing, and populating each device, with descriptions of common technical complications and advice for troubleshooting. Within this protocol are techniques for the consistent loading of small-volume NGM, the consistent drying of both the NGM and bacterial food, options for delivering pharmacological interventions, instructions for and practical limitations to reusing PDMS devices, and tips for minimizing desiccation, even in low-humidity environments. This technique allows the longitudinal monitoring of various physiological parameters, including stimulated activity, unstimulated activity, body size, movement geometry, healthspan, and survival, in an environment similar to the standard technique for group culture on solid media in Petri plates. This method is compatible with high-throughput data collection when used in conjunction with automated microscopy and analysis software. Finally, the limitations of this technique are discussed, as well as a comparison of this approach to a recently developed method that uses microtrays to culture isolated nematodes on solid media.

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.

多孔设备固体培养基上分离秀 丽隐杆线虫 的长期培养和监测

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, ...

使用多聚甲醛对 秀丽隐杆 线虫进行细菌代谢灭活

Methodology to Metabolically Inactivate Bacteria for Caenorhabditis elegans Research

Caenorhabditis elegans is a common model organism for research in genetics, development, aging, metabolism, and behavior. Because C. elegans consume a diet of live bacteria, the metabolic activity of their food source can confound experiments looking for the direct effects of various interventions on the worm. To avoid the confounding effects of bacterial metabolism, C. elegans researchers have used multiple methods to metabolically inactivate bacteria, including ultraviolet (UV)-irradiation, heat-killing, and antibiotics. UV treatment is relatively low-throughput and cannot be used in liquid culture because each plate must be examined for successful bacterial killing. A second treatment method, heat-killing, negatively affects the texture and nutritional quality of the bacteria, leading to the developmental arrest of C. elegans. Finally, antibiotic treatment can directly alter C. elegans physiology in addition to preventing bacterial growth. This manuscript describes an alternative method to metabolically inactivate bacteria using paraformaldehyde (PFA). PFA treatment cross-links proteins within bacterial cells to prevent metabolic activity while preserving cellular structure and nutritional content. This method is high-throughput and can be used in liquid culture or solid plates, as testing one plate of PFA-treated bacteria for growth validates the whole batch. Metabolic inactivation through PFA treatment can be used to eliminate the confounding effects of bacterial metabolism on studies of drug or metabolite supplementation, stress resistance, metabolomics, and behavior in C. elegans.

作者聚焦:使用多聚甲醛处理的细菌推进秀丽隐杆线虫研究 

The food source for Caenorhabditis elegans in the lab is live Escherichia coli. Since bacteria are metabolically active, they present a confounding variable in metabolic and drug studies in C. elegans. A detailed protocol to metabolically inactivate bacteria using paraformaldehyde is described here.

秀丽隐杆线虫研究的代谢灭活细菌的方法

Caenorhabditis elegans is a common model organism for research in genetics, development, aging, metabolism, and behavior. Because C. elegans consume a ...

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

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大规模混合阶段卡诺哈布迪蒂斯电子人种群的文化和分析

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

一

利用广域跟踪平台对大型线虫种群进行自动行为分析

培养

栽培

微生物分离株的高通量筛选对秀丽隐杆线虫健康的影响

在固体培养基上长期培养单个秀丽隐杆线虫，用于纵向荧光监测和厌恶干预

多孔设备固体培养基上分离秀丽隐杆线虫的长期培养和监测

秀丽隐杆线虫研究的代谢灭活细菌的方法

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

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大规模混合阶段 卡诺哈布迪蒂斯电子 人种群的文化和分析

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

一

利用广域跟踪平台对大型线虫种群进行自动行为分析

培养

栽培

微生物分离株的高通量筛选对 秀丽隐杆线虫 健康的影响

在固体培养基上长期培养单个 秀丽隐杆线虫 ，用于纵向荧光监测和厌恶干预

多孔设备固体培养基上分离秀 丽隐杆线虫 的长期培养和监测

秀丽隐杆线虫研究的代谢灭活细菌的方法

大规模混合阶段卡诺哈布迪蒂斯电子人种群的文化和分析

微生物分离株的高通量筛选对秀丽隐杆线虫健康的影响

在固体培养基上长期培养单个秀丽隐杆线虫，用于纵向荧光监测和厌恶干预

多孔设备固体培养基上分离秀丽隐杆线虫的长期培养和监测