我们感谢UAB的有针对性的代谢组学和蛋白质组学实验室，已支持部分由UAB的皮肤疾病研究中心（P30 AR050948 C. Elmets），UAB的加州大学圣地亚哥分校奥布莱恩急性肾损伤中心（P30 DK079337 A.阿加瓦尔）和UAB的肺部健康中心（R01，R01 HL110950 HL114439乙脑布莱洛克）。支持用于质谱仪是从一个，NCRR共享仪表授予（S10 RR19261 S.巴恩斯）。我们也感谢雷·摩尔的技术支持。C.线虫线虫遗传学中心，这是由NIH资助的菌株。这项工作得到由NIH（R01GM085105 MAM，包括ARRA行政补充）。

下文所述的协议被分为三个部分：蜗杆文化，前列腺素的提取，和前列腺素分析。正如所指出的，有多个点时，样品可以暂时存储在-80℃或-20℃。 1。蠕虫文化我们建议混合阶段全面LC-MS/MS蠕虫约6克。这应该提供足够的材料检测至少六个单独注射。对于定量分析的被称为PG的MRM在单次注射或两个1-2克就足够了。组成的一个特定的阶段的同步培养提取物的分析也是可能的。同时最多4个不同的菌株能够生长。例如，野生型控制和三个不同的突变株。 <ol><li>准备65 X大（16厘米）NGM板和细菌集中补充喂养。使用NA22细菌播种。为了使集中补充喂养，GRO细菌瓦特至少四个升NA22在LB培养基中16-24小时。自旋向下，除去上清液，并在100毫升M9的缓冲液中重悬细菌。 200〜400毫升的这种浓缩的细菌溶液可能需要为每个虫种。贮存于4℃ </li><li> 5 X-大板块开始蠕虫文化。蠕虫生长板约3〜4代，直到妊娠成人。应加入浓缩细菌板，以防止饥饿，必要时（〜1毫升/板材和更换前盖完全干燥）。 </li><li>妊娠雌雄同体洗净关闭板与M9的缓冲区，并收集在50毫升锥形聚丙烯管的蠕虫。 </li><li>允许妊娠雌雄同体“，以稳定的底部（约3-5分钟）。去除上清液，包括细菌，卵，幼虫阶段蠕虫。重悬在15毫升M9的缓冲区，轻轻混匀。 </li><li>蠕虫病毒悬液转移200微升每个60种子NGM板。分散在整个板块的蠕虫。保持蠕虫溶液混合板，直到所有被播种，以确保他们收到的蠕虫病毒的数量大致相等。 </li><li>补充急需的（〜1 ml/plate/12至24小时内），以防止饥饿的浓缩细菌。允许盖更换前板风干。 2-4代的种植蠕虫，取决于种子蠕虫原来的号码，或直到板妊娠成人。 </li><li>收获虫板应变时间。洗净蠕虫断板M9的缓冲区，并收集在50毫升聚丙烯管（ 如 6管）。使用M9的缓冲区去洗了个板，然后蠕虫填充缓冲区转移到下一盘。洗涤板数（ 如 10 日 ）后，转移到50毫升管的蠕虫填充的缓冲区。 M9的缓冲区填充管顶端。重复洗涤板的其余部分。 </li><li>随着妊娠的成年人稳定的底部在5-6分钟，取出上清液（约35毫升），并整合成一个或两个管。填充管50毫升FRESH M9缓冲液，静置3-5分钟，除去上清液离开妊娠大人。重复3次，或者，直到上清液是透明的。 </li><li>使用大口径巴斯德吸管，转让，许多蠕虫尽可能到两个15毫升聚丙烯锥形管。降速在1000 RCF（〜3,500 rpm时在CentraSL2），在临床离心5分钟。删除的上清和重复，直到所有的蠕虫被转让而在同一管沉淀。在-80℃下尽可能和存储蠕虫尽可能多的上清液中除去重复所有菌株。 </li></ol> 2。前列腺素的提取提取所需的试剂如下所示。该方法被修改的17 Golovko和Murphy，包括丙酮提取，然后通过液-液纯化，从而提高LC-MS/MS灵敏度。子弹搅拌机均质，虽然在过程中使用了类似的结果可以得到一个Dounce匀浆。叔丁基hydroxytolue的NE甲苯（BHT）和氮（N 2）下蒸发气体用于防止氧化。所有的玻璃器皿应硅化（Sigmacote）减少脂质结合。用有机溶剂提取应进行化学罩。 <ol><li>称取50毫升的聚丙烯锥形管。约6.0克冷冻蠕虫传输储存在-80℃下用热刀片附近的6.5毫升管贯穿从15毫升锥形管。可以使用的甲醇清洗刮勺以除去冷冻蠕虫颗粒从管的底部。通过切割通过沉淀的颗粒从顶部检索下部，密度较小的幼虫蠕虫和残留的细菌被留了下来。 </li><li>将预先称重的50毫升锥形管冷冻蠕虫。如果正在处理多个样品，使用相同的金额（6.0克）的组织进行比较研究。由于蠕虫解冻成糊状，添加或删除蠕虫用锅铲，以获得所需的质量。可选：广告D 1.00纳克PGF2α-D4标准，作为内部控制的提取效率。 </li><li> 0.005％BHT蠕虫病毒悬液中加入12毫升冰冷的2:1丙酮/盐水拌匀涡旋。蜗杆淤浆的1.5毫升的分液各12 5毫升自站立在子弹搅拌机用于塑料管。 </li><li>添加0.7-0.8毫升0.5毫米直径的二氧化铈稳定的氧化锆珠，每次5毫升管。关闭帽紧紧加载12管子弹搅拌机均质。搅拌机运行速度8-9的3分钟，然后把试管放在冰。请务必关闭镜头盖得很紧或内容可能洒出。从一个管检查10微升匀浆幻灯片上使用立体。应该很少有剩余的完整的蠕虫。重复一分钟以相同的速度，如果必要的。 </li><li>匀浆均匀地转移到四个10毫升锥形玻璃管使用9“巴斯德吸管避免传输珠子。清洗珠三管塞克ntially用1毫升1:2的丙酮/盐水含有BHT。其他管重复清洗，直到所有的珠子。传输剩余的溶液（〜4ml）中的10毫升玻璃试管中，并将其放置在冰上。 </li><li>在临床离心在约1,000 xg离心10分钟，在4℃下离心10毫升的玻璃管。从每管将上清液转移到一个干净的10毫升玻璃锥形管。 </li><li>在化学罩中，加入等体积的己烷，每次10毫升玻璃管。例如，添加3.5毫升丙酮/生理盐水溶液3.5 mL正己烷。以最大速度为30秒的涡流。 </li><li>在临床离心在约1,000 xg离心10分钟，在4℃下离心10毫升的玻璃管。丢弃上相，含有正己烷和中性电荷的脂质。 </li><li>酸化的下层相pH值至3.5，使用2 M甲酸（约100 - 150微升）。测试pH值用pH条。 </li><li>在每个试管中加入等体积的氯仿。涡最大速度为30秒，离心管在4°C INA临床离心机〜1,000 XG 10分钟。 </li><li>取出并丢弃上层水相。插入吸头通过任何残留的乳白色相间低级氯仿相，避免相间。收集下层相从4管中的每一个到一个干净的15毫升锥形玻璃管。在-20℃下放置至少24小时的孵育。在这一点上，其提取物可在-20℃下储存最多两个星期。 </li><li>以提取从冰箱中拿出，并丢弃其余的乳白色的水，如果目前顶层。氯仿相转移到聚四氟乙烯内衬标记的半德拉姆玻璃瓶采用了全新的巴斯德吸管。在化学罩，蒸发至干，将氯仿可溶组分下一个温柔流动的N 2气。将干燥的提取物可以储存在-20℃下长达2周。 PG提取和分析用于图1中所示的总体方案。 </li></ol> 3。前列腺素分析<ol><li>准备股票单独的参考，如前列腺素，前列腺素F2α或PGF 2α-d4上（1微克/毫升）的甲醇稀释，用甲醇：水（8:2体积/体积），以获得一个有效的解决方案的解决方案。准备了一系列的稀释液（100，10，1，0.1，0.01毫微克/毫升），以生成标准曲线。 </li><li>加入200微升甲醇：水（8:2体积/体积）C.干燥线虫脂质提取物（s）和调匀。如果小于6克蠕虫组织萃取，干燥的萃取物，再悬浮于甲醇：水溶液的量也少。 </li><li>准备流动相为0.1％甲酸的水[A]和含0.1％甲酸乙腈[B]。 </li><li>设置自动进样器在4℃，并在70支1.5毫升瓶架放置样品瓶。 </li><li>平衡协同水力RP-C18柱，0.1％甲酸水溶液，流速为0.2毫升/分钟约5分钟。 </li><li>入柱中注入样品（20〜50微升）。进行梯度洗脱启动使用10％B，去从0-11分钟11-14分钟80-100％B从80％B，返回到10％B在16分钟。总运行时间为20分钟。 13个标准品的保留时间，如表1所示，在参考文献3。 </li><li>介绍污水进入质谱仪使用负离子模式的的ESI接口工作在列。使用氮气作为雾化器，气帘气（CUR = 10）。碰撞气体，碰撞能量和温度都设定为10，-35 eV和600℃。去聚势（DP），碰撞能量（CE），池出口处电位（CXP），分别设定为-90，-35和-10。 </li><li>对于调查扫描（Q1的扫描），使用负离子模式下，对于大多数的PG（ 图2）的质量范围内的m / z 315-360。可以改变的范围内，这取决于化合物的质量（次）的利益。提取离子总离子流（TIC）的LC-MS离子色谱，并决定是否提取TED离子对应去质子化或加合物离子或碎片离子。 </li><li>对于MRM实验，大规模过渡的m / z三百一十一分之三百五十五的是，用于检测F1级的m / z一百九十三分之三百五十三用于F2类， 米/ z为 351/193或一百九十一分之三百五十一用于F3类（ 图3）。 MRM的，也可用于检测内标（例如， 米/ Z197分之357的PGF2α-d4上），并生成标准曲线。 Murphy 等附加信息PG质量转换18。 </li><li>对于MS / MS实验中，使用M / Z 355 F1级，M / Z的 F2 353for类和m / z 351for F3级的PG。C.线虫为F系列前列腺素的MS / MS数据，是可在图4和表2 3,16。 MS / MS数据的哺乳动物类二十烷酸是提供<a href="http://www.lipidmaps.org" target="_blank">www.lipidmaps.org</a> 。 </li><li>处理分析数据分析产品吨软件（1.4.2版，应用生物系统公司）。 </li></ol>

<ol>
	<li>Watts, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat+synthesis+and+adiposity+regulation+in+Caenorhabditis+elegans.">Fat synthesis and adiposity regulation in Caenorhabditis elegans.</a> Trends Endocrinol. Metab. 20, 58-65 (2009).</li><li>Marza, E., Lesa, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyunsaturated+fatty+acids+and+neurotransmission+in+Caenorhabditis+elegans.">Polyunsaturated fatty acids and neurotransmission in Caenorhabditis elegans.</a> Biochem. Soc. Trans. 34, 77-80 (2006).</li><li>Edmonds, J. W., 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=Insulin/FOXO+signaling+regulates+ovarian+prostaglandins+critical+for+reproduction.">Insulin/FOXO signaling regulates ovarian prostaglandins critical for reproduction.</a> Dev. Cell. 19, 858-871 (2010).</li><li>Kniazeva, M., Shen, H., Euler, T., Wang, C., Han, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+maternal+phospholipid+composition+and+IP(3)-dependent+embryonic+membrane+dynamics+by+a+specific+fatty+acid+metabolic+event+in+C.+elegans.">Regulation of maternal phospholipid composition and IP(3)-dependent embryonic membrane dynamics by a specific fatty acid metabolic event in C. elegans.</a> Genes Dev. 26 (3), 554-566 (2012).</li><li>Kosel, 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=Eicosanoid+formation+by+a+cytochrome+P450+isoform+expressed+in+the+pharynx+of+Caenorhabditis+elegans.">Eicosanoid formation by a cytochrome P450 isoform expressed in the pharynx of Caenorhabditis elegans.</a> Biochem. J. 435, 689-700 (2011).</li><li>Kubagawa, H. 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=Oocyte+signals+derived+from+polyunsaturated+fatty+acids+control+sperm+recruitment+in+vivo.">Oocyte signals derived from polyunsaturated fatty acids control sperm recruitment in vivo.</a> Nat Cell Biol. 8, 1143-1148 (2006).</li><li>Lucanic, 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=N-acylethanolamine+signalling+mediates+the+effect+of+diet+on+lifespan+in+Caenorhabditis+elegans.">N-acylethanolamine signalling mediates the effect of diet on lifespan in Caenorhabditis elegans.</a> Nature. 473, 226-229 (2011).</li><li>Gerisch, B., 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+bile+acid-like+steroid+modulates+Caenorhabditis+elegans+lifespan+through+nuclear+receptor+signaling.">A bile acid-like steroid modulates Caenorhabditis elegans lifespan through nuclear receptor signaling.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 5014-5019 (2007).</li><li>Edison, A. S. <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+pheromones+regulate+multiple+complex+behaviors.">Caenorhabditis elegans pheromones regulate multiple complex behaviors.</a> Curr. Opin. Neurobiol. 19, 378-388 (2009).</li><li>Funk, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandins+and+leukotrienes:+advances+in+eicosanoid+biology.">Prostaglandins and leukotrienes: advances in eicosanoid biology.</a> Science. 294, 1871-1875 (2001).</li><li>Wang, D., Dubois, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eicosanoids+and+cancer.">Eicosanoids and cancer.</a> Nat. Rev. Cancer. 10, 181-193 (2010).</li><li>Cha, Y. I., Solnica-Krezel, L., DuBois, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fishing+for+prostanoids:+deciphering+the+developmental+functions+of+cyclooxygenase-derived+prostaglandins.">Fishing for prostanoids: deciphering the developmental functions of cyclooxygenase-derived prostaglandins.</a> Dev. Biol. 289, 263-272 (2006).</li><li>Hata, A. N., Breyer, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+and+signaling+of+prostaglandin+receptors:+multiple+roles+in+inflammation+and+immune+modulation.">Pharmacology and signaling of prostaglandin receptors: multiple roles in inflammation and immune modulation.</a> Pharmacol. Ther. 103, 147-166 (2004).</li><li>Sugimoto, Y., Narumiya, S., Ichikawa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+function+of+prostanoid+receptors:+studies+from+knockout+mice.">Distribution and function of prostanoid receptors: studies from knockout mice.</a> Prog. Lipid Res. 39, 289-314 (2000).</li><li>Basu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoprostanes:+novel+bioactive+products+of+lipid+peroxidation.">Isoprostanes: novel bioactive products of lipid peroxidation.</a> Free Radic. Res. 38, 105-122 (2004).</li><li>Hoang, H. D., Prasain, J. K., Dorand, D., Miller, M. 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+heterogenous+mixture+of+F-series+prostaglandins+promotes+sperm+guidance+in+the+C.+elegans+reproductive+tract.">A heterogenous mixture of F-series prostaglandins promotes sperm guidance in the C. elegans reproductive tract.</a> PLoS Genetics. 9 (1), e1003271 (2013).</li><li>Golovko, M. Y., Murphy, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+LC-MS/MS+procedure+for+brain+prostanoid+analysis+using+brain+fixation+with+head-focused+microwave+irradiation+and+liquid-liquid+extraction.">An improved LC-MS/MS procedure for brain prostanoid analysis using brain fixation with head-focused microwave irradiation and liquid-liquid extraction.</a> J. Lipid Res. 49, 893-902 (2008).</li><li>Murphy, R. 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=Electrospray+ionization+and+tandem+mass+spectrometry+of+eicosanoids.">Electrospray ionization and tandem mass spectrometry of eicosanoids.</a> Anal. Biochem. 346, 1-42 (2005).</li><li>Milne, G. L., Yin, H., Morrow, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+biochemistry+of+the+isoprostane+pathway.">Human biochemistry of the isoprostane pathway.</a> J. Biol. Chem. 283, 15533-15537 (2008).</li><li>Lehtonen, M., Reisner, K., Auriola, S., Wong, G., Callaway, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass-spectrometric+identification+of+anandamide+and+2-arachidonoylglycerol+in+nematodes.">Mass-spectrometric identification of anandamide and 2-arachidonoylglycerol in nematodes.</a> Chem. Biodivers. 5, 2431-2441 (2008).</li><li>Kulas, J., Schmidt, C., Rothe, M., Schunck, W. H., Menzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+P450-dependent+metabolism+of+eicosapentaenoic+acid+in+the+nematode+Caenorhabditis+elegans.">Cytochrome P450-dependent metabolism of eicosapentaenoic acid in the nematode Caenorhabditis elegans.</a> Arch. Biochem. Biophys. 472, 65-75 (2008).</li><li>Watts, J. L., Browse, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+polyunsaturated+fatty+acid+synthesis+in+Caenorhabditis+elegans.">Genetic dissection of polyunsaturated fatty acid synthesis in Caenorhabditis elegans.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 5854-5859 (2002).</li></ol>

作者有没有利益冲突。

我们描述了一个程序类花生酸的提取和分析，重点对F系列的PG。有几个部分的程序，可能会产生问题。首先，重要的是该蠕虫的文化不挨饿，饥饿可以改变PG代谢。对于补充喂养，NA22细菌的建议，而不是更常用的OP50细菌，因为NA22达到更高的密度。然而，NA22菌株缺乏抗生素的耐药性，更容易受到污染。其次，蠕虫个人PG异构体合成，低丰度相对哺乳动物组织。这可能是部分原因是由于众多的异构体之间的冗余。我们建议约6克组织进行全面的分析和更强的信号。可以使用，如果较少的组织（1-2克）的干燥的萃取物再悬浮在更短的甲醇：水的溶液，以增加PG浓度。但是，只有一到两个注射剂可以被执行。我们的LC-MS/MS系统的检出限约10皮克PGF2α/毫升。密密麻麻的混合阶段蠕虫毫升（约1克）产生大约25-50皮克CePGF2。更敏感的质谱系统，可以减少分析所需的蜗杆组织的量。第三，样品PG提取效率之间的差异可以导致不同的结果。我们已经发现，提取效率是非常类似的，当组织中提取平行。要确定效率，加1.0纳克的PGF2α-D 4日作为内部控制的同质化步骤。 MRM 的m / z 357/197用于测量前列腺素F2α-d4上的浓度，相对于1 ng / ml的标准溶液，使用大量过渡。在提取过程中丢失的PGF2α-d 4中的量，然后计算。 可以改变的色谱参数和质量转变，我们包括检测其他的前列腺素和花生四烯酸。尽管有相当大的努力，我们一直无法识别D系列或E-SERIES PGS提取物中17。此外，我们并没有发现8 -异前列腺素F2α，8 -异PGE 2的自由基引发的过氧化反应，生成PG的立体异构体19是一种非选择性的混合物的特征。不知道是否蠕虫其他类型的PG合成。在C中已发现的内源性大麻素和各种环氧基和羟基代谢产物花生四烯酸和二十碳五酸线虫提取5,7,20,21。我们建议使用MRM和MS / MS相比正宗的定性和定量的标准，分别。小说类花生酸类物质，这些分析应结合脂肪的突变体，这是缺乏多不饱和脂肪酸合成酶22，以及一个功能检测，如果可能的话进行比较研究。分钟蠕虫中存在的多不饱和脂肪酸的数量是足够的PG合成分析脂肪的突变体是一个警告。例如， 脂肪-2（WA17）突变体的D12去饱和酶的活性有少量（野生型的22％〜5％），这些突变体仍能产生前列腺素6。 脂肪3（WA22）和脂肪-4（WA14），突变体不能合成的PG是来自于花生四烯酸和二十碳五酸16 。我们还发现， 脂肪的突变体通过上调PG合成的多不饱和脂肪酸类的损失，其余类补偿。例如，的脂肪-3（WA22）突变体没有大多数来自20个碳的多不饱和脂肪酸的PG的合成。相反，他们似乎上调小说PGS来自18碳不饱和脂肪酸16。到今天为止，我们还没有确定C.线虫应变PG的合成，是完全缺乏。这是可能的，前列腺素是必不可少的生长或发展。

前列腺素（PGs的）是一类重要的脂质激素已被广泛地研究，并在调节再生，免疫和发展在广泛的生物体12-14牵连。全面的系统生物学分析的PG非常适合，因为它们包含了一系列来自一个共同的前体（S）的血脂。线虫C.线虫遗传学和系统生物学来解决根本问题，是使用最广泛的模式生物之一。 我们已经证明，C。线虫综合F系列的PG，活动精子卵母细胞3,6,16指南。已经确定，三个，四个，五个双键组成的F1，F2，和F3类，分别从20个碳的多不饱和脂肪酸衍生的前列腺素F系列3,16。这些前列腺素合成独立环氧合酶尚未PGF1α和PGF2α立体的的，仍然GEnerated。 C.进行定性和定量分析线虫 PGS，LC-MS/MS是一个强大和敏感的分析技术。在执行这一分析之前，重要的是开发优化的提取方法，因为在分析过程的性能很大程度上依赖于提取物的质量。液相色谱法和质谱法只能提供预期的质荷比（M / Z），以及理想的峰形和保留时间为PG母离子。 C.前列腺素的代谢谱线虫是一项艰巨的任务，需要各种不同的MS收购策略。 LC-MS全扫描或调查扫描（Q1的扫描）确保，最电离的PG的生成质谱反应的优点。虽然调查扫描相对于野生型和突变型C.前列腺素的总分析提供有用的信息线虫 ，因轻微的前列腺素的检测灵敏度低。然而以下，液相色谱 - 质谱联用仪测量扫描可以给的全局视图的PG，用于分析预测影响代谢的一个大的PG人口的突变体是特别有用的。 在代谢物鉴定，LC-MS/MS分析化学合成PG标准是第一次演出，以取得他们的产品作为参考化合物离子光谱。这些光谱进行比较的一个未知的代谢物谱。多反应监测（MRM）模式用于增强的敏感性和特异性。 MRM实验中是通过指定母离子/产物离子质量的化合物过渡。通过了解的质量和目标分析物的结构，它是可以预测理论的MRM离子在很多未知的代谢物。 一个优化的LC-MS/MS方法分析异构前列腺素C.线虫尚未见报道。在这里，我们描述了一个提取和综合质谱法组成的LC-MS和LC-MS / MS方法进行检测，量化，并调查C.线虫 PG代谢产物。这种方法可以应用到其他的类二十烷酸。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>X-large (16 cm) plates</td>
			<td>Fisher Scientific</td>
			<td>0875714</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>E. coli strain NA22</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td>NA22</td>
			<td><a href="http://www.cgc.cbs.umn.edu" target="_blank">http://www.cgc.cbs.umn.edu</a></td>
		</tr>
		<tr>
			<td>Acetone, 399.5%</td>
			<td>Fisher Scientific</td>
			<td>A928-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Butylated Hydroxytoluene</td>
			<td>MP Biomedicals</td>
			<td>101162</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hexane, HPLC grade</td>
			<td>Fisher Scientific</td>
			<td>H302-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Formic acid, 399%</td>
			<td>Acros</td>
			<td>AC27048-0010</td>
			<td>Available through Fisher Scientific</td>
		</tr>
		<tr>
			<td>Chloroform, HPLC grade</td>
			<td>Acros</td>
			<td>AC26832-0010</td>
			<td>Available through Fisher Scientific</td>
		</tr>
		<tr>
			<td>PGF2&alpha;-d4</td>
			<td>Cayman Chemicals</td>
			<td>316010</td>
			<td>Cayman has a wide array of standards available for purchase</td>
		</tr>
		<tr>
			<td>Sterile screw cap self-standing 5 ml tube</td>
			<td>Fisher Scientific</td>
			<td>14-222-651</td>
			<td>For use with Bullet Blender</td>
		</tr>
		<tr>
			<td>0.5 mm zirconium oxide beads</td>
			<td>Next &gt;&gt;&gt; Advance</td>
			<td>ZrOB05</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 ml glass conical heavy-duty graduated centrifuge tubes</td>
			<td>Kimble Kimax</td>
			<td>45200-10</td>
			<td>Available through Fisher Scientific</td>
		</tr>
		<tr>
			<td>15 ml glass conical tubes</td>
			<td>Corning Pyrex</td>
			<td>8082-15</td>
			<td>Available through Fisher Scientific</td>
		</tr>
		<tr>
			<td>Sigmacote (Siliconizing reagent)</td>
			<td>Sigma-Aldrich</td>
			<td>SL2-100ML</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Teflon lined capped 1/2 Dram glass vial</td>
			<td>Fisher Scientific</td>
			<td>V1235C-TFE</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>CentraSL2 Clinical Centrifuge</td>
			<td>Thomas Scientific</td>
			<td>2517N92-TS</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bullet Blender 5 blue homogenizer</td>
			<td>Next &gt;&gt;&gt; Advance</td>
			<td>BBY5MB</td>
			<td>A 40 ml Dounce homogenizer can be used as an alternative</td>
		</tr>
		<tr>
			<td>Synergi 4 &mu;m Hydro-RP 80 &Aring; LC Column 250 x 2.0 mm</td>
			<td>Phenomenenex</td>
			<td>00G-4375-B0</td>
			<td>HPLC Column</td>
		</tr>
		<tr>
			<td>SecurityGuard Cartridges AQ C18 4 x 2.0 mm</td>
			<td>Phenomenenex</td>
			<td>AJ0-7510</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SecurityGuard Guard Cartridges Kit</td>
			<td>Phenomenenex</td>
			<td>KJ0-4282</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Shimadzu Prominence HPLC with refrigerated auto sampler</td>
			<td>Shimadzu Scientific Instruments, Inc.</td>
			<td>&nbsp;</td>
			<td>Any standard HPLC system coupled to a triple quadrupole mass spectrometer should be sufficient</td>
		</tr>
		<tr>
			<td>API 4000 triple quadrupole mass spectrometer</td>
			<td>Applied Biosystems/MDS SCIEX</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>
			<table border="1">
				<tbody>
					<tr>
						<td colspan="2">M9 buffer recipe (4 liter)</td>
					</tr>
					<tr>
						<td>12 g</td>
						<td>KH2PO4</td>
					</tr>
					<tr>
						<td>24 g</td>
						<td>Na2HPO4</td>
					</tr>
					<tr>
						<td>20 g</td>
						<td>NaCl</td>
					</tr>
					<tr>
						<td>Up to 4 liter</td>
						<td>Deionized water</td>
					</tr>
					<tr>
						<td colspan="2">Aliquot to eight 1 liter bottles. Autoclave and cool.</td>
					</tr>
					<tr>
						<td>0.5 ml/bottle</td>
						<td>1 M MgSO4</td>
					</tr>
				</tbody>
			</table>
			</td>
		</tr>
	</tbody>
</table>

prostaglandin extraction and analysis in caenorhabditis elegans

线虫正在成为一个强大的动物模型来研究生物学脂质1-9。前列腺素是一类重要的类二十烷酸，是脂质信号是来自于多不饱和脂肪酸（PUFAs）10-14。这些信号分子是很难研究，因为他们的低丰度和反应性。前列腺素的特征是环戊烷环结构的位于脂肪酸骨干内。在哺乳动物中，前列腺素可以通过环加氧酶的酶-依赖性和非依赖性通路10,15形成。C.线虫独立环氧合酶6,16,17广泛的前列腺素合成。一个大F系列前列腺素类已经确定，但花生酸类物质的研究是在早期阶段有足够的空间，新的发现。在这里，我们描述了一个过程，前列腺素和其他类花生酸类物质的提取和分析。带电脂质s的提取从大量的蠕虫病毒培养，采用液 - 液萃取技术，液相色谱 - 电喷雾电离串联质谱（LC-ESI-MS/MS）分析。氘化的前列腺素类似物，如前列腺素F 2α-d 4中的夹杂物，作为内标，建议进行定量分析。可以使用多反应监测或MRM量化和比较野生型和突变型的动物之间的特定类型的前列腺素。碰撞诱导分解或MS / MS可以使用以获得有关的重要结构特征。液相色谱-质谱（LC-MS）调查选定的质量范围的扫描，如M / Z 315-360可以用来评估全球变化中的前列腺素水平。我们提供所有三个分析的例子。这些方法将为研究人员提供一个工具集，用于发现新的花生四烯酸，其代谢途径及划定。

样品制备进行了改编自Golovko和墨菲17液-液萃取。它提供了出色的内部标准（PGF2αD4）恢复。从C PG提取的总体方案图1所示的线虫 。色谱条件进行了优化，提供&gt; 30类花生酸的标准（ 表1显示了13个标准）的基线分离。水力RP柱（250×2.0 mm内径）含0.1％甲酸的水和乙腈提供了最好的分离和灵敏度。调查突变提取野生型和脂肪3所示图2笔全球级离子扫描质量范围为315-360个原子质量单位内缺乏20碳不饱和脂肪酸的脂肪（WA22）突变体。 F3类PG是突出。在图3中 ，野生型提取物的MRM分析显示多个PG异构体的F1（ 图3A </stro<html>纳克&gt;）（ 图3B），F2和F3（ 图3C和3D）类。要么两个质量过渡，可以检测F3类的m / z 351/193或M / Z一百九十一分之三百五十一。 CePGF2主要包括的PGF2α对映体。 CePGF1可能是PGF1α或它的对映体。 图4示出了碰撞诱导分解CePGF2（RT = 11.8）相比，PGF2α标准（RT = 11.8）。产物离子细微的差别可能是由于低的丰度CePGF2和合作洗脱化合物中提取。 <img alt="图1" src="/files/ftp_upload/50447/50447fig1.jpg" /> 图1。 C.示意图线虫的 PGS提取和LC-MS分析 。请注意，丙酮/盐水和氯仿0.005％BHT，以尽量减少脂质氧化。有关详细信息，请参阅文本。https://www.jove.com/files/ftp_upload/50447/50447fig1large.jpg“目标=”_blank“&gt;点击这里查看大图。 <img alt="图2" fo:content-width="4.75in" fo:src="/files/ftp_upload/50447/50447fig2highres.jpg" src="/files/ftp_upload/50447/50447fig2.jpg" /> 图2。 M / Z 315-360野生型和突变体的脂肪-3（WA22）提取。液相色谱保留时间（RT）的调查扫描显示面板上的[A]。提取离子RT = 11.3显示野生型[B]的脂肪-3（WA22）[C]提取。 PG质量的m / z 351的F3类被高亮显示。 CPS，计数/秒;阿木，原子质量单位。 <img alt="图3" fo:content-width="4.25in" fo:src="/files/ftp_upload/50447/50447fig3highres.jpg" src="/files/ftp_upload/50447/50447fig3.jpg" /> 无花果由图3。 MRM分析野生型的提取物。F1级的PG检测与质量过渡的m / z 355/311 [A]。请注意，一些疏水性化合物（RT&gt; 14分钟），这是不太可能的PG，也检测到这种过渡。 F2类前列腺素检测与群众过渡的m / z 353/193 [B]。 F3类前列腺素的检测为质量过渡的m / z193分之351[C] 或 m / Z191分之351[D]。请注意，提取液中含有多个PG的每个类的同分异构体。数字对应于表2中所示的前列腺素。 CePGF2的浓度是1.8毫微克/毫升。 RT，滞留时间; CPS，计数/秒。 <img alt="图4" fo:content-width="5.5in" fo:src="/files/ftp_upload/50447/50447fig4highres.jpg" src="/files/ftp_upload/50447/50447fig4.jpg" /> 图4。碰撞诱导分解的化学合成esized PGF2α和CePGF2的在面板上的箭头[A]表示PGF2α和CePGF2的（RT = 11.8）之间共享的产品或碎片离子。的商品在M / Z = 309 和 m / z为 193的离子产生的指示的裂解位点（突出显示的a和 b），对应于所示的结构，特征F系列的PG [B]。 CPS，计数/秒。 <a href="https://www.jove.com/files/ftp_upload/50447/50447fig4large.jpg" target="_blank">点击这里查看大图</a> 。 <table border="1" fo:keep-together.within-page="always"><tbody><tr><td> 标准 </td><td> RT（分钟） </td><td> [MH] - M / Z </td><td> 主要产品离子MS / MS </td></tr><tr><td> 20 -羟基PGE 2的</td><td> 9.43 </td><td> 367 </td><td> 349，331，287，234，189，129，109 </td></tr&gt; <tr><td> PGF 3 - </td><td> 11.26 </td><td> 351 </td><td> 333，307，289，271，245，219，209，193，191，171，165，111 </td></tr><tr><td> 血栓素B2 </td><td> 11.66 </td><td> 369 </td><td> 289，191，177，169，151 </td></tr><tr><td> PGF 2 - </td><td> 11.80 </td><td> 353 </td><td> 335，309，291，273，263，247，235，209，193，171，165，111 </td></tr><tr><td> PGF 1 - </td><td> 11.79 </td><td> 355 </td><td> 319311 337，301，293，275，265，237，211，195 </td></tr><tr><td>脂氧素B 4 </td><td> 12.38 </td><td> 351 </td><td> 201，191189，165，155，115，107，71，59 </td></tr><tr><td> PGD ​​2 </td><td> 12.56 </td><td> 351 </td><td> 315 271203 189 </td></tr><tr><td> 5（S），6 -脂氧素A 4（R） </td><td> 12.83 </td><td> 351 </td><td> 235，217，189，144，135，115，99，59 </td></tr><tr><td> PGA 2 </td><td> 14.02 </td><td> 333 </td><td> 315，297，271，235，191，189，175，163，137，113，109 </td></tr><tr><td> Δ12-PGJ 2 </td><td> 14.20 </td><td> 333 </td><td> 271，189，123 </td></tr><tr><td>白三烯B 4 </td><td> 14.73 </td><td> 335 </td><td> 181，109，93，71，69，59，57 </td></tr><tr><td> 15D-Δ12,14-PGJ 2 </td><td> 16.80 </td><td> 315 </td><td> 297，271，217，203，158 </td></tr><tr><td> 5（S）-HPETE </td><td> 17.88 </td><td> 335 </td><td> 97，83，81，57 </td></tr></tbody></table> 表1中。保留时间和13类花生酸LC-MS/MS方法 3 标准的主要产物离子 。 超过30标准，使用发展LC-MS/MS程序的。不同的色谱保留时间（RT），即使在非常相似的前列腺素。一个例外是PGF1α和PGF2α。然而，这些PGS的istinguished通过母离子的质量（[MH] - M / Z）和碰撞诱导分解光谱（MS / MS）。 <table border="1" fo:keep-together.within-page="always"><tbody><tr><td> 前列腺素类 </td><td> 在图3中的编号 </td><td> [MH] - M / Z </td><td> 主要产品离子MS / MS </td></tr><tr><td> F1（CePGF1） </td><td> 1 </td><td> 355 </td><td> 319，301，311，293，275，265，237，223，211，195，157 </td></tr><tr><td> F1 </td><td> 2 </td><td> 355 </td><td> 311，301，293，275，265，211，195，167，157 </td></tr><tr><td> F2（CePGF2） </td><td> 3 </td><td> 353 </td><td> 309，291，273，263，247，209，193，171，165，127 </td></tr><tr><td> F2 </td><td> 4 </td><td> 353 </td><td> 309，291，273，263，255，247，219，209，193，171，113 </td></tr><tr><td> F3 </td><td> 5 </td><td> 351 </td><td> 315，307，289，275，249，205，193，191，167，153，139 </td></tr><tr><td> F3 </td><td> 6 </td><td> 351 </td><td> 333，289，271，261，245，223，193，191，163 </td></tr></tbody></table> 表2中。几个主要C.碰撞诱导分解产物离子线虫 F1，F2，和F3的PG如图3所示 。其他较丰富的异构体的MS / MS将不会显示。这些数据是从多个分析，并非所有产品离子可能会看到在给定运行。鉴于提取物的复杂性，一些不太丰富的产品离子可能来自另一名家长离子具有类似的保留时间或结果的干扰。峰2是有可能的立体异构体CePGF1，峰4是有可能的立体异构体CePGF2。埃德蒙兹等人 （2010年），其他数据3。

Watch this Scientific Journal Video about Prostaglandin Extraction and Analysis in Caenorhabditis elegans at JoVE.com

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

在本文中，我们描述了一个优化的过程，前列腺素等类花生酸类物质的提取和分析<em&gt; C。线虫</em利用LC-MS/MS。

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, ...

prostaglandin-extraction-and-analysis-in-caenorhabditis-elegans

Research

JoVE Journal

Biology

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

19.4K 次观看.  University of Alabama at Birmingham. 在本文中，我们描述了一个优化的过程，前列腺素等类花生酸类物质的提取和分析 C。线虫

视频: Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

分离和<em在体外激活线虫精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

科研

生物学

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

实心板为基础的饮食限制在线虫</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

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 ...

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 ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

线虫的核苷酸和蛋白质提取联合

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

线虫的耗氧率测定

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying 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 ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

建模年龄相关的神经退行性疾病在 卡诺哈布迪蒂斯埃利根

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...