我们想感谢他的宝贵帮助医生安东尼米兰达Vizuete。我们也想表达我们的宝贵的技术援助表示感谢萨尔玛Boulayoune和伊莎贝尔卡瓦列罗。这项工作是由军政府安达卢西亚（BIO - 272）的赠款资助。目前在欧洲的遗传实验的法律协议，这项研究已经进行了。

1：渗透避免检测。 <ol><li>检测前约16-24小时，挑到一个新的NGM板含有OP50 大肠杆菌 L4各基因型的幼虫阶段动物大肠杆菌 andincubate在20 ° C第二天，开始实验青壮年。 </li><li>建议开展“盲目”的检测。每个被检测的应变板应relabelled第二个实验者，使用的实验已经完成时，将东窗事发relabelled板进行检测。 </li><li>检测的一天，准备与4M原液的果糖，1％的刚果红溶液在室温下完全溶解。我们建议检查的解决方案之前，每个检测以来，染料可能随着时间的沉淀。 </li><li> NGM板的环形圈（直径为1厘米的）概述了固体培养基的中心，红色4M果糖溶液与15μL。让果糖溶液浸泡成的琼脂，这通常需要2至5分钟。 </li><li>广场内的每个环的应变个人年轻的成年动物，并在随后的10分钟，以确定渗透屏障的响应。避免了环比在连续的6倍多的动物被列为正常;尝试在不到半年的退出环被认为是在渗透压的敏感性缺陷。 注意 ：必须在每个检测控制应变。 N2年轻的成年动物作为阳性对照，因为他们果断地避免环屏障。已饿死，通过的dauer幼虫或过于干燥的板未来的动物，不应该使用。在开始的时候，控制动物应重复进行评估，以确认检测板和解决方案是正确的。 </li></ol> 2：产生击倒蠕虫通过RNAi喂养。 <ol><li> RNAi的板块：NGM的RNA干扰喂养板每公升含有17克琼脂，2.5克蛋白胨，1毫升5毫克毫升-1胆固醇3.0 g氯化钠;填补烧瓶与H 2 O和高压灭菌器，以1公升。后琼脂冷却到约65℃，加入25毫升1个M KPO 4，pH值6.0，1毫升的1 M 氯化钙 ，1毫升1M硫酸镁4，0,5毫升羧（50毫克毫升-1）和1毫升1M IPTG的。如果您正在准备扎实NGM开始，由微波熔化固体培养基，随后放置在板凳上冷却，然后添加如前所述，羧KPO 4，pH值6.0， 氯化钙 ， 硫酸镁和IPTG液体NGM。 每60毫米的培养皿中加入10毫升NGM。允许冷却，并保持在室温下过夜，使用前翻转板。板可以储存在4 ° C，约4-5天，在一个塑料袋。 </li><li>细菌的制备及诱导： 大肠杆菌 HT115（DE3）菌株分离的殖民地，L4440包含一个相应的靶基因片段的载体转化到卢里亚Bertani（LB）琼脂平板（17克琼脂，蛋白胨10克，5克，酵母提取物，每公升10克氯化钠）含有氨苄青霉素（50微克/毫升）和四环素（15微克/毫升）。 选择一个细菌菌落和50μg/ mL氨苄青霉素的LB接种，并成长为6 8 H晃动在37 ° C，种子上的准备NGM板这种文化的下降，并彻底晾干后方可被孵化过夜板（ 12月24日H）在室温下，让细菌的生长，并开始上岗。在黑暗中孵化，因为四环素对光敏感，可能会影响板之间的RNAi实验的可变性。 它是需要使用一个积极的和一个用于RNAi喂养实验阴性对照。阳性对照是大肠杆菌 HT115细胞转化与L4440包含UNC - 22的基因序列的载体。 UNC - 22基因敲除产生“抽搐表型”。阴性对照与L4440空载体转化的大肠杆菌HT115细胞。 </li><li> L4从接种到NGM板OP50无细菌，并培育与20 NGM板阶段蠕虫蠕虫的处理和计分：第一天，地点° 12小时C（空腹）。 第二天，转移到与特定目标基因的RNAi表达的细菌seedded板年轻的禁食成人雌雄同体。保留40-48小时，在20 ° C至产生F1子代。 到与相同的细菌接种另一盘，然后把几个F1成虫。 24-48小时后，选择和隔离从F2代，年轻的成年人（完全形成突起外阴几个鸡蛋）和表型的得分。 注 ：在神经细胞的RNAi诱导有一定的局限性，因为在C线虫的神经系统难治性属性的 RNAi。为了克服这种低效率的问题，在神经元，它是推荐使用的RRF - 3 株神经元10，过敏背景的RNAi的影响。在我们的实验没有differencES之间的布里斯托尔N2和RRF - 3株被发现的基因和表型分析。 </li></ol> 3：使用菌株。 c. 在在这项工作中使用的线虫株见表1。突变株，越过对N2的野生 -至少四次，以去除不受欢迎的可能已在诱变协议产生的随机突变类型。 OP50 发送菌株 suppliied 线虫遗传中心，美国明尼苏达大学，HT115（DE3） 大肠杆菌质粒携带NRX - 1 pL4440（JA：C29A12.5）和NLG - 1（JA：C40C9.5彼得Askjaer博士，炫酷Andaluz Biología DEL Desarrollo（公共天线），中船重工大学巴勃罗Olavide，塞维利亚，西班牙）的基因片段。 4：代表结果。 说明性的结果表示在图1和2。十大动物，每一株和最低的三个副本实验进行。 <img alt="图1" src="/files/ftp_upload/1616/1616Fig1.jpg"/> 图1。实验渗透避税行为。 对照菌株和NRX - 1（ok1649和tm1961）V缺乏mutantworms回应向后倒车时，他们遇到一个渗透屏障（4M果糖）。NLG - 1（ok259和tm474）X突变体无法检测到这一障碍。在缺乏NLG - 1和NRX - 1菌株CRR21（ok259 ok1649）VX和CRR23（tm1961; ok259）;双突变体VX恢复野生型表型。 *表示由T -学生测试的显着性差异（P≤0.001），在每个应变的响应布里斯托尔N2的野生型，T -学生的考试<img alt="图2" src="/files/ftp_upload/1616/1616Fig2.jpg"/> 图2。实验通过RNAi喂养击倒蠕虫。 HT115（DE3） 大肠杆菌转化空pL4440载体或含有相应的目标NRX - 1或NLG - 1被用来喂不同的蠕虫病毒株的基因片段。 *表示T -学生的考试空pL4440载体美联储的N2应变比较pL4440载体携带一个目标NLG - 1基因片段的细菌美联储的N2应变响应的显着性差异（P≤0.001 ）， 。

<ol>
	<li>Sudhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 455 (7215), 903-903 (2008).</li><li>Fabrichny, I. P., Leone, P., Sulzenbacher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 56 (6), 979-979 (2007).</li><li>Garber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Science. 317 (5835), 190-190 (2007).</li><li>Wang, K., Zhang, H., Ma, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 459 (7246), 528-528 (2009).</li><li>Ward, S., Thomson, N., White, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Journal of comparative neurology. 160 (3), 313-313 (1975).</li><li>Bargmann, C. I., Thomas, J. H., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+Spring+Harbor+symposia+on+quantitative+biology.">Cold Spring Harbor symposia on quantitative biology.</a> 55, 529-529 (1990).</li><li>White, J. G., Southgate, E., Thomsom, J. N., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Philos. Trans. R. Soc. Lond. B Biol. Sci. 314, 1-1 (1986).</li><li>Culotti, J. G., Russell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genetics. 90 (2), 243-243 (1978).</li><li>Fire, A., Xu, S., Montgomery, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 391 (6669), 806-806 (1998).</li><li>Timmons, L., Fire, 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> Nature. 395 (6705), 854-854 (1998).</li><li>Simmer, F., Tijsterman, M., Parrish, S., Koushika, S. P., Nonet, M. L., Fire, A., Ahringer, J., Plasterk, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Curr Biol. 12, 1317-1317 (2002).</li><li>Missler, M., Zhang, W., Rohlmann, 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> Nature. 423 (6943), 939-939 (2003).</li><li>Varoqueaux, F., Aramuni, G., Rawson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 51 (6), 741-741 (2006).</li><li>Graf, E. R., Zhang, X., Jin, S. X., Scheiffele, P., Fan, J., Choih, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell. 119 (7), 1013-1013 (2004).</li><li>Scheiffele, P., Fan, J., Choih, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell. 101 (6), 657-657 (2000).</li><li>Jamain, S., Quach, H., Betancur, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature genetics. 34 (1), 27-27 (2003).</li><li>Szatmari, P., Paterson, A. D., Zwaigenbaum , L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature genetics. 39 (3), 319-319 (2007).</li></ol>

Neurexins和neuroligins执行突触传递的11和12个突触连接的分化中的重要作用。两种分子已被确定为自闭症13,14的候选基因。 在这个视频中，我们展示一个简单的方法，它可以让我们研究的影响渗透在C回避反应的基因的作用线虫 。的neuroligin缺陷的突变体缺陷检测渗透强度，但缺乏突变体在neurexin显示野生型菌株类似的响应，避免一个4M的果糖?...

表1。C.线虫株。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 应变 </td><td> 基因 </td><td> 等位基因 </td><td> 来源 </td></tr><tr><td>布里斯托尔氮气</td><td> - </td><td> - </td><td> ACGC </td></tr><tr><td> VC228 </td><td> NLG - 1 </td><td> ok259 </td><td>政府总部大楼</td></tr><tr><td> FX00474 </td><td> NLG - 1 </td><td> tm474 </td><td> bNBP - JAPAN </td></tr><tr><td> VC1416 </td><td> NRX - 1 </td><td> ok1649 </td><td>政府总部大楼</td></tr><tr><td> FX1961 </td><td> NRX - 1 </td><td> tm1961 </td><td> NBP - JAPAN </td></tr><tr><td> NL2099 </td><td> RRF - 3 </td><td> pk1436 </td><td>政府总部大楼</td></tr><tr><td> CRR21 </td><td> NRX - 1 NLG - 1; </td><td> ok1649; ok259 </td><td>这项工作</td></tr><tr><td> CRR22 </td><td> NRX - 1 NLG - 1; </td><td> tm1961; ok259 </td><td>这项工作</td></tr></tbody></table> A. 线虫遗传中心，美国明尼苏达大学。 B.国家实验动物“的线虫C. elegans的 ”生物资源项目。 日本东京女子医科大学。

osmotic avoidance in caenorhabditis elegans: synaptic function of two genes, orthologues of human nrxn1 and nlgn1, as candidates for autism

Neurexins和neuroligins目前在兴奋和抑制性突触细胞粘附分子，他们需要正确的神经网络功能 1 。这些蛋白在突触前和突触后膜2。在小鼠的研究表明，neurexins和neurologins在突触传递 1中的重要作用。最近的报告表明，改变神经元连接在人体神经系统的的发展可能构成的自闭症谱系障碍的许多案件3的病因的基础上。 线虫可以用来作为实验工具，方便的突触组成部分的运作的研究，因为它简单实验室试验，并得到了充分的特点，其神经系统和突触接线。 在 C 线虫NRX - 1和NLG - 1基因是同源NRXN1和NLGN1人类基因编码α- neurexin - 1和的neuroligin - 1蛋白，分别的。在人类与线虫，组织neurexins和neuroligins是类似的功能域。 线虫的头部包含amphid，线虫，介导的反应不同的刺激，包括渗透强度的感觉器官。 amphid 12纤毛树突和突触前终端轴突4感官双极神经元。这些神经元命名ASHR和ASHL，两个特别重要的渗透的感觉功能，检测与高渗透压实力 5水溶性的驱虫剂。这两种神经元的树突延长到嘴的尖端和轴突延伸到神经环，在那里他们作出决定的行为反应 6的其他神经元突触连接。 为了评估在高渗透压的力量避免neurexin和的neuroligin的影响，我们展示了不同的反应NRX - 1和NLG - 1基因缺陷的线虫突变体，使用一个4M的果糖环7一种方法。行为表型被证实使用特定的RNAi 克隆 8 。 在 C 线虫 ，触发的RNAi的dsRNA可管理喂养9。通过食物传递的dsRNA诱导的利益，从而使遗传组件和网络途径的识别基因的RNAi干扰。

Watch this Scientific Journal Video about Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism at JoVE.com

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins和neuroligins执行突触分化和传输中的重要作用的神经元膜的粘附蛋白。缺乏突变体的neuroligin<em&gt; C。线虫</em&gt;检测渗透强度的缺陷，但它们也包含在编码neurexin基因的突变时，他们恢复野生型表型。

渗透避免IN线虫：突触功能的两个基因，人类同源 NRXN1 NLGN1，作为候选人自闭症

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network ...

osmotic-avoidance-caenorhabditis-elegans-synaptic-function-two-genes

Research

JoVE Journal

Biology

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

11.7K 次观看.  Facultad de Ciencias, Universidad de Córdoba. Neurexins和neuroligins执行突触分化和传输中的重要作用的神经元膜的粘附蛋白。缺乏突变体的neuroligin C。线虫检测渗透强度的缺陷，但它们也包含在编码neurexin基因的突变时，他们恢复野生型表型。

视频: Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

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

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 ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

生化和高通量的微观评估的脂肪含量<em&gt;秀丽隐杆线虫</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using 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, ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

多不饱和脂肪酸的膳食补充剂<em&gt;秀丽隐杆线虫</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

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 ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

透射电镜下体的样品制备与成像

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

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 ...