星号（*）表示新的或修改的步骤相比，Christensen 等人 1 1。材质设置<ol><li>在细胞培养过程需要大量的鸡蛋从妊娠的成年人中分离的。 C.成长线虫对8P琼脂平板NA22（可通过线虫遗传协会-长城电脑）接种细菌分离大量鸡蛋。在这些板用蛋白胨的量为8倍，通常是用于NGM板的量。较高的蛋白胨浓度更有效地维系着NA22细菌的生长，这出乎OP50-，生长在厚层。 </li></ol> 8P板的配方: 将3克氯化钠，20g的细菌用胰蛋白胨，1升无菌蒸馏水和高压釜30分钟25克琼脂。让介质凉爽，在55℃，然后加入无菌过滤的1毫升胆固醇（5毫克/毫升的EtOH），将1ml 1的MgCl 2的1毫升硫酸镁和25毫升KP缓冲液（500毫升储:5克K 2 HPO 4，30克KH 2 PO 4，pH值6.00）。倒液琼脂培养基中成10毫米培养皿中（25毫升/盘）。 <ol start="2"><li>第二天散布各富含蛋白胨琼脂板的整个表面上用1毫升NA22 E的在2XYT培养基（16克胰蛋白胨，10克酵母提取物，5克NaCl的1升的无菌水，pH 7.0）中于37℃培养的大肠杆菌过夜这些细菌构成了丰富的食物来源，会形成一层厚厚的支持大量妊娠成人的生长。离开种子板在室温下过夜，使细菌的生长。该过程可以通过在37℃下进行4-5小时来加速。 </li><li>饿死的动物转移到播种8P板。清洗过的动物用5-6毫升M9的缓冲区或水的匮乏NGM板加1-2毫升此悬液，以每8P板。 </li><li>允许生长的动物和乘法UNT金正日板是由妊娠成人人口在汇合。 </li><li>隔离要求下编制的蛋线虫用5-6的裂解溶液胚胎细胞，从妊娠的成年人。 </li></ol>裂解液配方 5毫升新鲜的漂白剂1.25毫升10 N的NaOH和18.5毫升的无菌H 2 O的该混合物必须准备每次使用前新鲜。 <ol start="6"><li>从洗净用鸡蛋缓冲妊娠成人孤立的蛋: </li></ol>鸡蛋缓冲配方 118 mM氯化钠，48毫米氯化钾，2毫米氯化钙2，2毫米氯化镁2，25毫米HEPES，pH值7.3，渗透压340毫渗量。 <ol start="7"><li>删除与几丁质酶处理周围的鸡蛋蛋壳。几丁质酶是在酸性pH值25最高活性的酶。在蛋缓冲液pH 6.5溶解几丁质酶（Sigma公司，目录号C6137）在2毫克/毫升的最终浓度。存储几丁质酶股票溶液在-20℃下在1ml等份在无菌的15毫升锥形管中。等分试样可被储存在-20℃至几个月。 </li><li> C.成长线虫培养细胞上覆盖着花生凝集素灭菌盖玻片（直径12 mm）。溶解花生植物血凝素在无菌水（0.5毫克/毫升）。花生凝集素溶液不应该过滤或高压灭菌。它也并不需要被用UV光处理。存储2毫升分装在-20°C可保存6个月。 </li><li>完整的细胞培养液（500毫升）中包含500个ML L-15培养基购自Gibco，将50ml胎牛血清（热灭活），7.7克蔗糖（45毫渗量），5毫升100微克100 U / mL青霉素和/毫升链霉素（2％）。在完全培养基中，然后用0.20微米的微孔过滤器过滤。 </li></ol>注意的是，蛋缓冲器和培养基分别具有340摩尔渗透压浓度和345毫渗透摩尔浓度。事实上，相反的哺乳动物细胞，C。线虫细胞中有相对高的渗透压需要准备的解决方案，在将要与所述细胞的细胞膜直接接触时，必须考虑到计数。这些试剂的配方进行了调整，以达到所需的渗透压，这是用一个渗压计1测量。这是没有必要使用一个渗透压力计如果这些配方是完全按照与护理用于制备这些试剂。然而，人们应该使用osmoter如果其他解决方案需要做好准备，他们的食谱这里不报，或在任何使用C的出版物线虫培养细胞。 2。鸡蛋隔离<ol><li>我们建议开始与至少四个8P板收集到足够的鸡蛋12井培养细胞（24孔板）。 </li><li>开始在手术前解冻的花生凝集素原液一管。置于高压灭菌的盖玻片在孔的底部的24孔板，加入200μl的花生凝集素的盖玻片。孵育1小时或uNTIL细胞准备进行电镀。完全取出花生凝集素和用1毫升无菌水灭菌一次洗井。彻底去除盖玻片花生凝集素是避免细胞聚集是必不可少的。 </li><li>洗妊娠关闭成人用无菌水高压灭菌的琼脂平板。收集悬浮成两个无菌锥形50ml管中。留在冰上的管中长达5分钟，以允许蠕虫沉淀在管（*）的底部。与转印塑料吸管除去水，并将其与新鲜无菌水高压灭菌取代。通过台式离心分离，在200×g离心（〜1200 rpm）离心10分钟（*）沉淀蠕虫。重复该最后步骤至少为3。 </li><li>传送蠕虫到无菌的15毫升锥形管中，沉淀它们在200×g离心（〜1200 rpm）离心10分钟（*）。不使用更高的离心速度，以避免收集细菌在管的底部，以及。有时候，离心后的虫还没有完全沉淀。 AF三每一清洗和去除之前上清液，将管5分钟冰上。在冷却水的蠕虫沉淀到管的底部。 </li><li>去除水中加5-6毫升裂解液（见材料设置）。摇动该悬浮液轻轻地5-10分钟，然后开始监控虫溶胞体视显微镜下，每2-3分钟。一滴悬浮液也可以放置在盖玻片，以方便检查。孵化时间取决于漂白剂的新鲜度;买小瓶装的漂白剂，每月开新瓶。 </li><li>当〜蠕虫的70-80％被溶解（从孵化开始10分钟），停止裂解反应中加入9mL的蛋缓冲液pH 7.3（见材料设置）。离心该悬浮液在200×g离心（〜1200 rpm）离心10分钟。从这点上有一个本生灯上，在板凳上，以防止再次污染的鸡蛋（*）的。 </li><li>小心取出用无菌塑料移液上清，洗涤沉淀3-4x，其中蛋缓冲区中，直到溶液澄清。确保每次洗涤过程中混合颗粒以及在蛋缓冲区。 </li><li>鸡蛋是从用30％蔗糖溶液的动物尸体分离。重悬浮颗粒在2毫升无菌鸡蛋缓冲和加入2ml 60％的蔗糖溶液（股票在无菌鸡蛋缓冲区）。拌匀，离心20分钟在200 XG（〜1200转）（*）。 </li><li>小心地从离心分离机取出试管。将卵悬浮在溶液的上方。使用P1000移液器和无菌针头，转移所有鸡蛋放入一个新的无菌的15 mL锥形管。 </li><li>加入10毫升无菌蛋缓冲到管和离心机在200×g离心（〜1200 rpm）离心10分钟。重复洗涤3×。请确保在鸡蛋缓冲区每次清洗时将鸡蛋完全悬浮。 </li></ol> 3。胚胎干细胞解离进行下一步骤的程序使用的是层流罩的无菌条件下进行。虽然动物袍菌上板，将洗涤液并用含漂白剂的裂解溶液处理，如果不是所有的细菌应消除大部分。因此，使用在层流罩在这一点上的程序可以防止新的卵悬浮液的污染。 <ol><li>重悬沉淀鸡蛋1毫升2毫克/毫升的几丁质酶（股票鸡蛋缓冲液pH 6.5（*）），并将它们转移到一个新的无菌的15 mL锥形管。摇动该管，在室温下10-30分钟。根据酶和房间的温度的新鲜度的精确的温育时间的变化，因此应为每个准备确定。建议在开始监视孵育10分钟后倒置细胞培养显微镜下的蛋。注:根据我们的经验，低pH值增加几丁质酶活性。为此，我们使用蛋缓冲液，pH 6.5以溶解几丁质酶（配方上面的报道，其中pH用NaOH调整至6.5）。 </li><li>时〜80％的蛋壳是由消化几丁质酶处理（ 图1 AB），通过离心分离沉淀的鸡蛋，在900×g离心（〜2500 rpm）离心3分钟（*）。使用P1000移液器和无菌针头，小心取出上清液，加3毫升的L-15培养基（*）。 </li><li>传送鸡蛋成直径6厘米的板，并用10毫升无菌注射器配备有18号针头轻轻地解离的细胞。将一滴悬浮液到一个新的塑料培养皿中，并通过在显微镜下观察监测解离度。在此过程中不要吸入空气进入注射器，以避免损坏电池。继续解离，直到〜80％的细胞解离。 </li><li>使用无菌5微米的微孔过滤器过滤悬浮液。细胞悬浮液必须经过过滤，以除去细胞团块，未消化的卵和孵出的幼虫。通过过滤器过滤额外4-5毫升新鲜的L-15介质恢复所有的细胞。不要使用在过滤步骤用力过猛，以免达maging过滤器和/或细胞。 </li></ol> 4。细胞培养<ol><li>通过离心分离沉淀的分离的细胞在900×g离心（〜2500 rpm）离心3分钟（*）。使用P1000移液器和无菌针头小心地取出所有上清。重悬细胞沉淀在完整的L-15培养基和板1毫升/。加入培养基中的量依赖于所用的8P板的数量，在平板上蠕虫的汇合处，以及将要在细胞上进行的实验的类型。细胞密度可以使用血细胞计数器确定。对于膜片钳记录电镀〜230000个细胞/ cm 2为最佳浓度。 </li><li>保持24孔板中含有湿纸巾，避免培养基蒸发特百惠塑料容器。存储容器，在湿润的培养箱中在20℃和环境空气。 </li><li>这些细胞通常是准备在24小时内试验时的形态分化和快递的绿色荧光蛋白标记物的离子是完整的。细胞可以保存在文化长达2周，但它们通常是最健康的长达7-9天电镀后。介质需要更换，每天一次，以保持健康的细胞。 </li></ol>

线虫是一种功能强大的模式生物破译参与发展，行为和衰老的遗传途径。其方便茎主要从难易程度可以遗传操作，并从它的循环寿命短。尽管它的便利性，C.线虫有其局限性。C.线虫细胞是微小的，在加压的角质层，限制了需要直接访问的细胞，例如电生理和药理学技术，或特定类型的细胞的分离，如通过微阵列分析的基因表达图谱方法的应用局限。该三线虫的细胞培?...

秀丽隐杆线虫 （ 线虫 ）是一款功能强大的模式生物研究细胞功能，分化和行为的分子基础。虽然它的基因组，代谢和生物合成途径类似于脊椎动物"，它的遗传和分子可追踪性都远远大于2。在它的优点是它的外形和简洁的解剖，其快速的生命周期（3天在25°C），短寿命（2周）和大量的后代（&gt; 200）。由于其雌雄同体的性质和生命周期短，分子和基因操作是直接在C。线虫 ，包括转基因动物3,4和诸如RNA干扰5基因敲低技术的应用的生成。C.线虫体和蛋壳是透明的。因此，细胞可以容易地可视化在成人和使用标准显微镜的胚胎。在过去的40年里，C。线虫 </em&gt;的社区已经为下创建的宝贵资源线虫的研究，包括收集了大量的突变体，基因敲除和转基因，解剖和发展6,7的详细说明，包括完整的重建神经系统8，和一个完全测序的基因组这是很好的说明，并将提供给整个社会（ <a href="http://www.wormbase.com" target="_blank">www.wormbase.com</a> ）。 尽管有许多优点，一些实验方法已在C挑战线虫 。这些包括需要访问到组织或细胞类型的细胞和分离的质膜的那些。事实上Ç。线虫组织都在其加压流体静骨架，这是不容易通过酶处理或洗涤剂消化密闭。在20世纪90年代末仪Goodman和珍妮特里士满率先为C的电生理记录的方法线虫神经元和肌肉细胞中原位 9,10。虽然这些方法给了我们重要的见解和神经元在体内的肌肉功能，它们是具有挑战性和低吞吐量。替代的方法来研究细胞功能的体内已经制定了，大多尤其是在使用基因编码的钙传感器，如GCamP和卡默莱昂11-13 体内钙成像。这些方法虽然不允许使用的药理学工具，因为它们是应用在完整的活的动物。 在培养C.第一次尝试线虫细胞在体外大规模被他的博士论文14的制备过程中由莱尔德布卢姆。不幸的是，难以与细胞到衬底的粘附性差遇到，不良细胞的分化和存活无法建立此早期协议作为一个强大的细胞培养方法。 1995年，埃德加和他的同事发表的程序由孤立的单个C和文化来研究细胞分化和形态发生。线虫胚胎15。通过用酶处理和手动解离的组合蛋壳的消化获得的胚胎干细胞，继续增殖，产生高达〜500个细胞15。随后，梁先生和同事培养一个小数字卵裂球的研究肠道形态。它们表明，1 体外分离ë卵裂球产生由通过心尖粘着连接16彼此进行交互创建了一个结构类似于肠腔偏振肠细胞。布氏及其同事还报道了培养℃的类似的方法。线虫胚胎细胞在体外 17。 在此基础上的早期作品，克里斯滕森和同事开发出一种强大的协议培养胚胎Ç。线虫细胞在体外 1。他们表明分离出来的C。线虫细胞可以分化成各种细胞类型和保持它们在体内具有的功能，包括细胞特异性标志物的表达。那些在体内具有挑战性的几种技术，可以在隔离C.应用线虫胚胎细胞。这些包括电1,18 19，成像和免疫化学技术20,21，以及通过荧光激活细胞的特定细胞类型分选（FACS）对细胞特异性cDNA文库22,23的结构的隔离。如RNA干扰（RNAi）的基因敲除技术可以对培养C.施加线虫细胞1，用叠氮基糖作为糖蛋白发现的一个工具了一种新的代谢标记方法最近已在体外培养的C.开发线虫细胞24。 总之，细胞培养方法扩展数组Ø可应用到的C. f技术线虫模型，以努力破译基因功能的生物体的情况下。我们在这里描述的协议C.培养线虫在体外 ，这在很大程度上是基于最早由克里斯蒂安森和他的同事1中所述的协议胚胎细胞。

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a method for culturing embryonic c. elegans cells

线虫是一种强大的模型系统中，其中的遗传和分子生物学技术是很容易适用的。直到最近，虽然，需要直接访问到细胞和特定的细胞类型的隔离技术，无法在C应用线虫 。这种限制是由于这一事实，即组织内被加压的角质层这是不容易通过与酶和/或洗涤剂处理消化的局限。基于早期的先驱工作，莱尔德布鲁姆，克里斯滕森和同事1开发出一个强大的方法培养C.线虫胚胎干细胞在大规模。鸡蛋是从妊娠成人分离与漂白/ NaOH处理，随后用壳多糖酶处理以除去蛋壳。胚胎细胞，然后通过手动移液解离，并铺板于血清中丰富的介质基片覆盖的玻璃。在隔离牢房的24小时开始改变形态和表达细胞特异性马克分化RS。C.线虫细胞用这种方法培养的存活最多2周体外和已用于电生理，免疫和成像分析以及它们已被分类并用于微阵列分析。

线虫培养的细胞分化并表达细胞特异性标志物 Christensen和使用台盼蓝染色的同事证明，&gt; 99％的胚胎C的线虫细胞存活的分离过程。在9日和镀后22，分别为85％和65％，仍健在1。孤立的胚胎C.线虫的细胞必须坚持的衬底，以区分。细胞没有附着形成团块和它是否生存是不明确的。细胞的分化开始2-3小时电镀后并持续至24小时。 24小时内，细胞承?...

Watch this Scientific Journal Video about A Method for Culturing Embryonic C. elegans Cells at JoVE.com

A Method for Culturing Embryonic C. elegans Cells

我们在这里描述的修订协议，用于胚胎C.大型文化线虫的细胞。 C.胚胎线虫使用这种方法的细胞在体外培养时，出现分化和概括的基因的表达在细胞中的具体方式。需要直接访问到细胞中，或从其它组织中的特定细胞类型的分离技术可以对C.施加线虫培养细胞。

一种用于培养胚胎<em&gt; C。线虫</em&gt;细胞

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

a-method-for-culturing-embryonic-c-elegans-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

A Method for Culturing Embryonic C. elegans Cells

15.5K 次观看.  University of Miami. 我们在这里描述的修订协议，用于胚胎C.大型文化线虫的细胞。 C.胚胎线虫使用这种方法的细胞在体外培养时，出现分化和概括的基因的表达在细胞中的具体方式。需要直接访问到细胞中，或从其它组织中的特定细胞类型的分离技术可以对C.施加线虫培养细胞。 

视频: A Method for Culturing Embryonic C. elegans Cells

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

科研

生物化学

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

突触荧光自动定量<em&gt; C。线虫</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

神经科学

C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining

The C. elegans germline makes an excellent model for studying meiosis, in part due to the ease of conducting cytological analyses on dissected animals. Whole mount preparations preserve the structure of meiotic nuclei, and importantly, each gonad arm contains all stages of meiosis, organized in a temporal-spatial progression that makes it easy to identify nuclei at different stages. Adult hermaphrodites have two gonad arms, each organized as a closed tube with proliferating germline stem cells at the distal closed end and cellularized oocytes at the proximal open end, which join in the center at the uterus. Dissection releases one or both gonad arms from the body cavity, allowing the entirety of meiosis to be visualized. Here, a common protocol for immunofluorescence against a protein of interest is presented, followed by DAPI staining to mark all chromosomes. Young adults are immobilized in levamisole and quickly dissected using two syringe needles. After germline extrusion, the sample is fixed before undergoing a freeze crack in liquid nitrogen, which helps permeabilize the cuticle and other tissues. The sample can then be dehydrated in ethanol, rehydrated, and incubated with primary and secondary antibodies. DAPI is added to the sample in the mounting medium, which allows reliable visualization of DNA and makes it easy to find animals to image under a fluorescent microscope. This technique is readily adopted by those familiar with handling C. elegans after a few hours spent practicing the dissection method itself. This protocol has been taught to high-schoolers and undergraduates working in a research lab and incorporated into a course-based undergraduate research experience at a liberal arts college.

C. elegans Gonad Dissection and Freeze Crack for Immunofluorescence and DAPI Staining

This is a commonly used method for C. elegans gonad dissection followed by freeze crack, which produces germline samples for immunofluorescence via antibody staining, or for simple DAPI staining to visualize DNA. This protocol has been successful for undergraduates in a research lab and in a course-based undergraduate research experience.

秀丽隐杆线虫 用于免疫荧光和DAPI染色的性腺解剖和冷冻裂缝

The C. elegans germline makes an excellent model for studying meiosis, in part due to the ease of conducting cytological analyses on dissected animals. ...

遗传学

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

可视化的神经细胞胞质分裂过程中<em&gt; C。线虫</em&gt;胚胎

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

新颖的指标来表征线虫胚胎伸长率<em&gt;秀丽隐杆线虫</em

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

高通量筛选和生物传感与荧光 C。线虫菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

生物学

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

测量细菌的影响<em&gt; C。线虫</em&gt;行为使用一个鸡蛋保留含量

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

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 ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

研究卡埃纳哈布迪炎细胞结构和细胞形态学的定量方法

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

C. elegans Maintenance

Ceanorhabditis elegans has been, and is still, used to great success as a model organism for studying a variety of developmental, genetic, molecular and even physical phenomena. In order to use C. elegans to its full potential, proper care and attention to the basic maintenance of this powerful organism is essential.
In this video you will learn the basic housing and feeding requirements of C. elegans, how to correctly handle and manipulate worms using a worm pick and how to freeze and recover important worm stocks. Towards the end of the video we will visit a few applications of modifying the housing, feeding and manipulation of these important animals.

Maintenance of Caenorhabditis elegans

秀丽隐杆线虫的实验室基本培养

Ceanorhabditis elegans has been, and is still, used to great success as a model organism for studying a variety of developmental, genetic, molecular and ...

一种用于培养胚胎

摘要

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此视频中的章节

一种简单的高通量化学筛选方法在秀丽线虫中的应用

突触荧光自动定量

秀丽隐杆线虫用于免疫荧光和DAPI染色的性腺解剖和冷冻裂缝

可视化的神经细胞胞质分裂过程中

新颖的指标来表征线虫胚胎伸长率

高通量筛选和生物传感与荧光 C。线虫菌株

测量细菌的影响

培养

研究卡埃纳哈布迪炎细胞结构和细胞形态学的定量方法

秀丽隐杆线虫的实验室基本培养

一种用于培养胚胎

摘要

探索更多视频

此视频中的章节

一种简单的高通量化学筛选方法在秀丽线虫中的应用

突触荧光自动定量

秀丽隐杆线虫 用于免疫荧光和DAPI染色的性腺解剖和冷冻裂缝

可视化的神经细胞胞质分裂过程中

新颖的指标来表征线虫胚胎伸长率

高通量筛选和生物传感与荧光 C。线虫菌株

测量细菌的影响

培养

研究卡埃纳哈布迪炎细胞结构和细胞形态学的定量方法

秀丽隐杆线虫的实验室基本培养

秀丽隐杆线虫用于免疫荧光和DAPI染色的性腺解剖和冷冻裂缝