这项工作是由支持的新研究员格兰特[2014R1A1A1005553]韩国国家研究基金会（NRF）为JIL;和延世大学未来之星挑战格兰特[2015-22-0133]为JIL

1.准备NGT-3D和NGB-3D解决方案<ol><li>准备以下的无菌溶液:1升的0.1454摩尔NaCl溶液中，1升的1M的CaCl 2，1升的1M 硫酸镁的，LB培养基（LB），1升1M的KPO 4缓冲液（108.3克KH 2的PO 4和35.6克K 2的HPO 4，并填写H 2 O操作1升）。利用这些解决方案NGM的生产可以在先前的协议10被发现。 </li><li>在121℃，15分钟的高压釜的解决方案。 </li><li>准备为50ml灭菌的5毫克/毫升胆固醇溶液。在50毫升的锥形管，混合0.25克胆固醇和50毫升99.99％乙醇拌匀。 不要高压 。使用50毫升注射器用0.45μm针筒式滤器灭菌胆固醇溶液中。这也将消除任何不溶解胆固醇。 </li><li> （可选）准备的10ml 2&#39;-脱氧-5-氟尿苷（的FUdR）股票150毫米的。在一个15毫升的锥形管，混合0.3693克的的FUdR和10ml蒸馏水。摇匀。 </li></ol> 2.准备细菌培养为NGT-3D和NGB-3D <ol><li>接种10毫升LB培养基细菌。用于线虫喂食标准细菌大肠杆菌菌株OP50。 </li><li>培养在37℃下在振荡培养箱中过夜的细菌接种。 </li><li>在用于连续稀释制备，等分9毫升的NaCl溶液到无菌的15毫升锥形管中。例如，分装9毫升共打入7管做一个10-7稀释OP50应变大肠杆菌 。 </li><li>使用无菌1000微升吸管，吸管将1ml细菌培养或稀释的细菌培养成育新管9毫升氯化钠溶液，涡旋10毫升混合好。重复，直到达到期望的细菌稀释。 注意:所希望的细菌稀释取决于细菌的种类和条件以及确切的实验条件。例如，一个10 -6 - 10 -8稀释OP50菌株大肠杆菌的足以从1到生产-每6.5毫升NGT-3D管200的细菌菌落。蠕虫可靠发达国家和正常再生时的菌落数超过60，其在10 -6的稀释发生- 10-7。对于NGB-3D，使用10 -8稀释。 </li></ol> 3.使NGT-3D和NGB-3D（200ml）中<ol><li>制备细菌培养后，混合0.6克氯化钠，1g的造粒琼脂，和在一个500ml烧瓶0.5克胨。插入一个磁性搅拌棒的烧瓶中。 </li><li>添加195毫升蒸馏水和用铝箔覆盖该烧瓶的口。 </li><li>高压釜121℃15分钟。 </li><li>热高压灭菌烧瓶放置到一个搅拌盘，并以中等速度搅拌至少2小时。冷却烧瓶至40℃。一定要充分冷却从这里在高温下持续可能导致成品琼脂混浊。但是，较低的温度可能会导致在琼脂过早硬化。 <ol><li> （可选）要加快冷却过程，将烧瓶在40℃水浴15分钟，放置在一个搅拌盘前。 </li></ol></li><li>当琼脂培养基的温度达到40℃时，加入200微升的1M的CaCl 2，200微升5毫克/毫升胆固醇溶液，200微升的1M MgSO 4上和5ml 1M的KPO 4缓冲液作为溶液继续搅拌至1mM的CaCl 2，5微克/毫升胆固醇，1mM的MgSO 4上，1mM的KPO 4的最终浓度。 <ol><li> （可选）NGT-3D寿命测定添加80μl的150mM的的FUdR的到120微米12的最终浓度。 </li></ol></li><li>加6毫升10毫升稀释后直接从步骤2.4细菌培养成瓶的。 <ol><li> （可选）对于NGT-3D，删除6毫升琼脂培养基步骤3.6之前保持温暖分开。此介质将用于细菌 - 自由顶层。 </li></ol></li><li>使用无菌的程序，分配所述媒体到无菌的培养室。确保盒盖紧紧关闭。 <ol><li> （可选的）为了防止细菌菌落从琼脂的顶部表面上形成，从步骤媒体3.7完全硬化之前在顶部从步骤3.6.1使无菌琼脂培养基的层。这将在NGT-3D的顶部创建一个无菌区。 </li><li>为NGT-3D，倾6.5毫升介质插入8ml上述透明塑料试管，使细菌琼脂层，并小心地分配在半硬化3D媒体的顶部200微升无菌介质，以使薄无细菌在上面自由层。 </li><li>对于NGB-3D，倒65毫升媒体到25 平方厘米的透明塑料细胞培养瓶。这一数额应填写25 平方厘米的细胞培养瓶的瓶体。 </li></ol></li><li>离开垂直于室温室一周以允许细菌菌落生长到至少1毫米直径的具有相当规模。 <ol><li> （可选）对于NGT-3D寿命试验，用铝箔盖室，以防止的FUdR的光降解。 </li></ol></li></ol>在NGT-3D（相对育雏面积测定）蠕虫人口4.测量健身<ol><li>挑选使用铂丝的L4阶段蠕虫挑一个无细菌的NGM板转移。允许蠕虫自由走动几分钟，以去除附在它身上的细菌。 </li><li>重复步骤4.1和确保该蠕虫病毒是从细菌免费的。一般来说，4.1两个重复就足够了。 </li><li>小心地将清洁蜗杆与铂丝挑3D媒体的表面上。该蠕虫应最终进入琼脂进入3D琼脂矩阵。 </li><li>关上盖子松松地让一些空气进入管道，但防止琼脂培养基的干燥。 </li><li>在20℃孵育96小时。 </li><li> 4天之后，紧紧地关闭盖子和放置NGT-3D培养室分成一个88℃的水浴以熔化的琼脂。热杀死虫子，但他们的身体保持不变。 注意:使用8个毫升管的NGT-3D，20 - 30分钟潜伏期通常是足够的。 </li><li>用玻璃吸管，转移熔化的媒体到9厘米塑料培养皿。塑料吸管不建议，因为蠕虫病毒往往可以坚持给他们。 </li><li>使用透射立体声解剖显微镜，计数在L3，L4蠕虫和成年阶段的数目。不要指望那些L2级和年轻的蠕虫，作为F1和F2代可以在这里混淆。因此，这个实验是一个相对的育雏规模试验，而不是总育雏规模试验。 </li></ol> 5.图像和NGB-3D记录蠕虫行为<ol><li>使用铂丝挑L4期蠕虫挑选并通过转移到一个无菌NGM板并使其自由几分钟移动除去粘在表面上的任何细菌。 </li><li>重复步骤5.1，以确保禾RM是细菌免费的。 </li><li>小心地将清洁蠕虫到NGB-3D的瓶用铂丝拾取的颈部附近的琼脂表面的中心。该蠕虫应最终进入琼脂进入3D琼脂矩阵。 </li><li>关上盖子松松地让一些空气进入管道，同时防止琼脂培养基的干燥。 </li><li>图片或录制立体声传输解剖显微镜下的蠕虫病毒。调整焦距为蠕虫移动通过3D矩阵。 </li></ol>

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The authors have nothing to disclose.

采用经典的线虫的生长介质板线虫的实验室培养是至关重要的该研究线虫提供了数百个重要的发现。在这里，我们提出新的方法来培养线虫中更准确地反映其三维天然栖息地的环境。尽管其它方法已被用于在3D 13观察线虫 ，这是第一个协议，它允许蠕虫培养在固体三维基质。这里展示的两种方法，NGT-3D和NGB-3D，让科学家问3D养身健体，发展壮大的问题，也是图像...

The study of the nematode Caenorhabditis elegans in the laboratory has led to seminal discoveries in the field of biology over the last five decades1. C. elegans was the first multicellular organism to have its genome sequenced in 19982, and it has been invaluable in understanding the contributions of individual genes to the development, physiology, and behavior of a whole organism. Scientists now are looking to further understand how these genes may contribute to the survival and reproductive fitness of organisms in their natural environments, asking questions about ecology and evolution at the genetic level3-5. 
C. elegans once again can provide an excellent system to answer these questions. However, little is known about C. elegans biology in natural nematode habitats, and there are no current methods to simulate controlled natural conditions of C. elegans in the laboratory. In the lab, C. elegans is cultivated on the surface of agar plates seeded with E. coli bacteria6. In nature, however, C. elegans and related nematodes can be found sparsely inhabiting soils throughout the globe, but they are specifically found thriving in rotting fruits and vegetative matter7,8. These three-dimensional (3D) complex environments are quite different from the simple 2D environments to which worms are exposed to in the laboratory. 
To begin to answer questions about the biology of nematodes in a more natural 3D setting, we have designed a 3D habitat for laboratory cultivation of nematodes we called Nematode Growth Tube 3D or NGT-3D for short9. The goal was to design a 3D growth system that allows for comparable growth, development, and fertility to the standard 2D Nematode Growth Media (NGM) plates10. This system supports the growth of bacteria and nematodes over their entire life cycles in 3D, allows worms to move and behave freely in three dimensions, and is easy and inexpensive to manufacture and employ. 
In the current study, we provide a step-by-step method to manufacture NGT-3D and evaluate worm development and fertility. In addition to assessing worm fitness in 3D, we sought to image, video, and assess worm behavior and physiology in 3D cultivation. Thus, in addition to NGT-3D, we present here an alternate method called Nematode Growth Bottle 3D or NGB-3D, for the microscopic imaging of C. elegans during 3D cultivation. This will be especially important for the study of known behaviors identified in 2D, and the identification of novel behaviors unique to 3D cultivation.

<table><tbody><tr><td>LB broth, Miller (Luria-Bertani)</td><td>Difco</td><td>224620</td><td></td></tr><tr><td>Sodium chloride</td><td>DAEJUNG</td><td>7548-4400</td><td>58.44 MW</td></tr><tr><td>Agar, Granulated</td><td>Difco</td><td>214530</td><td></td></tr><tr><td>Peptone</td><td>Bacto</td><td>211677</td><td></td></tr><tr><td>Calcium chloride, dihydrate</td><td>Bio Basic</td><td>CD0050</td><td>2*H&lt;sub&gt;2&lt;/sub&gt;O; 147.02 MW</td></tr><tr><td>Cholesterol</td><td>Bio Basic</td><td>CD0122</td><td>386.67 MW</td></tr><tr><td>Ethyl alcohol</td><td>B&amp;amp;J</td><td>RP090-1</td><td>99.99%; 46.07 MW</td></tr><tr><td>Magnesium sulfate, anhydrous</td><td>Bio Basic</td><td>MN1988</td><td>120.37 MW</td></tr><tr><td>Potassium phosphate, monobasic, anhydrous</td><td>Bio Basic</td><td>PB0445</td><td>136.09 MW</td></tr><tr><td>2&#039;-Deoxy-5-fluorouridine</td><td>Tokyo Chemical Industry</td><td>D2235</td><td>246.19 MW</td></tr><tr><td>Potassium phosphate, dibasic, anhydrous</td><td>Bio Basic</td><td>PB0447</td><td>174.18 MW</td></tr><tr><td>Multi-Purpose Test Tubes</td><td>Stockwell Scientific</td><td>ST.8570</td><td>8 ml</td></tr><tr><td>Test Tube Closures</td><td>Stockwell Scientific</td><td>ST.8575</td><td></td></tr><tr><td>Cell Culture Flask</td><td>SPL Lifescience</td><td>70125</td><td>25 cm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Research Stereo Microscope</td><td>Nikon</td><td>SMZ18</td><td></td></tr><tr><td>High-Definition Color Camera Head</td><td>Nikon</td><td>DS-Fi2</td><td></td></tr><tr><td>PC-Based Control Unit</td><td>Nikon</td><td>DS-U3</td><td></td></tr><tr><td>NIS-Elements Basic Research, Microscope Imaging Software</td><td>Nikon</td><td>MQS32000</td><td></td></tr></tbody></table>

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.

NGT-3D的结构是一个简单的和直接的协议，其导致在整个琼脂（ 图1A）间隔开小的细菌菌落在琼脂填充试管。蠕虫可通过琼脂矩阵自由移动，寻找和消费的细菌菌落。为了证实线虫是否可以复制和NGT-3D正常生长，我们比较土壤肥力与3D幼虫发育与标准的2D NGM板。在育雏相对大小分析，成年线虫在NGT-3D雌雄同体只是重现，以及在标准的2D NGM板雌雄同?...

Watch this Scientific Journal Video about Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory at JoVE.com

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

我们提出了一个简单的方法来构建三维线虫培养系统称为NGT-3D和NGB-3D。这些可以被用来研究在更类似于天然线虫栖息比标准2D实验室线虫培养板栖息线虫健身和行为。

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 ...

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Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

17.7K 次观看.  Yonsei University. 我们提出了一个简单的方法来构建三维线虫培养系统称为NGT-3D和NGB-3D。这些可以被用来研究在更类似于天然线虫栖息比标准2D实验室线虫培养板栖息线虫健身和行为。 

视频: Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

科研

遗传学

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

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

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

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

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

生物化学

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans has emerged as one of the best models for studying development. From an optical point of view, its size and transparent body make this nematode an ideal specimen for DIC (Differential Interference Contrast or Nomarski) microscopy. This article illustrates a protocol for growing C. elegans nematodes, preparing and mounting their embryos, performing 4D microscopy and cell lineage tracing. The method is based on multifocal time-lapse records of Nomarski images and analysis with specific software. This technique reveals embryonic developmental dynamics at the cellular level. Any embryonic defect in mutants, such as problems in spindle orientation, cell migration, apoptosis or cell fate specification, can be efficiently detected and scored. Virtually every single cell of the embryo can be followed up to the moment the embryo begins to move. Tracing the complete cell lineage of a C. elegans embryo by 4D DIC microscopy is laborious, but the use of specific software greatly facilitates this task. In addition, this technique is easy to implement in the lab. 4D microscopy is a versatile tool and opens the possibility of performing an unparalleled analysis of embryonic development.

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

Here, we present a protocol for preparing and mounting Caenorhabditis elegans embryos, recording development under a 4D microscope and tracing cell lineage.

4D显微镜:使用Nomarski显微镜揭示 秀丽隐杆线虫 胚胎发育

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans ...

发育生物学

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 ...

生物学

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

用于秀丽隐杆线虫长期生长和成像的简单微流控芯片

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

生物工程

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

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 ...

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 ...

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

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

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

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

在固体培养基上长期培养单个 秀丽隐杆线虫 ，用于纵向荧光监测和厌恶干预

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

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

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

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

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

多孔设备固体培养基上分离秀 丽隐杆线虫 的长期培养和监测

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

栽培

摘要

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此视频中的章节

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

4D显微镜:使用Nomarski显微镜揭示秀丽隐杆线虫胚胎发育

基本的

用于秀丽隐杆线虫长期生长和成像的简单微流控芯片

一种用于培养胚胎

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

在固体培养基上长期培养单个秀丽隐杆线虫，用于纵向荧光监测和厌恶干预

多孔设备固体培养基上分离秀丽隐杆线虫的长期培养和监测

栽培

摘要

探索更多视频

此视频中的章节

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

4D显微镜:使用Nomarski显微镜揭示 秀丽隐杆线虫 胚胎发育

基本的

用于秀丽隐杆线虫长期生长和成像的简单微流控芯片

一种用于培养胚胎

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

在固体培养基上长期培养单个 秀丽隐杆线虫 ，用于纵向荧光监测和厌恶干预

多孔设备固体培养基上分离秀 丽隐杆线虫 的长期培养和监测

4D显微镜:使用Nomarski显微镜揭示秀丽隐杆线虫胚胎发育

在固体培养基上长期培养单个秀丽隐杆线虫，用于纵向荧光监测和厌恶干预

多孔设备固体培养基上分离秀丽隐杆线虫的长期培养和监测