Name: cultivation of caenorhabditis elegans in three dimensions in the laboratory
Uploaded: 2016-12-12T14:00:00.000+00:00
Duration: 9 min 1 s
Description: Watch this Scientific Journal Video about Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory at JoVE.com

この作品は、韓国国立研究財団（NRF）からJILへの新しい研究者グラント【2014R1A1A1005553]によってサポートされていました。 [2015-22-0133] JILへと延世大学の未来のリーダーチャレンジグラント

1. NGT-3DとNGB-3Dのためのソリューションを準備<ol><li> 0.1454 M NaCl溶液1 L、1 MのCaCl 2、1MのMgSO 4、溶原性ブロス（LB）、KH 2の1 M KPO 4緩衝液（108.3グラムの1 Lの1 Lの1 L：以下の無菌溶液を準備しますK 2 HPO 4のPO 4 35.6 gであり、1 LにH 2 Oを充填します）。これらのソリューションを使用してNGMの生産は以前のプロトコル10で見つけることができます。 </li><li> 121℃、15分のオートクレーブソリューション。 </li><li>無菌の5mg / mlのコレステロール溶液50mlを準備します。 50ミリリットルの円錐管では、コレステロールの0.25グラムと99.99％のエタノール50mlを混合し、よく混ぜます。 オートクレーブしないでください 。 0.45μmのシリンジフィルターで50mlの注射器を用いてコレステロール溶液を滅菌します。これはまた、任意の溶解していないコレステロールを除去します。 </li><li> 2&#39;-デオキシ-5-フルオロ（FUDR）株式の150 mMのの10ミリリットルを用意（オプション）。 15ミリリットルコニカルチューブでは、0.3693グラムを混ぜますFUDRおよび蒸留水10mlを。よくまぜろ。 </li></ol> 2. NGT-3DとNGB-3Dのための細菌培養を準備<ol><li>細菌と10mlのLBブロスに接種。 C.エレガンス供給のために使用される標準的な細菌は、 大腸菌株OP50です。 </li><li>文化一晩振とう培養器で37℃で細菌接種。 </li><li>連続希釈の準備のために、滅菌した15ミリリットルコニカルチューブにNaCl溶液のアリコート9ミリリットル。例えば、7チューブの合計に一定分量9mlをOP50株大腸菌の10 -7希釈を行います。 </li><li> NaCl溶液の9ミリリットルで、無菌の新しいチューブに無菌千μlのピペット、ピペット1mlの細菌培養物または希釈した細菌培養物を使用して、よく10ミリリットルの混合物をボルテックス。目的の細菌の希釈が達成されるまで繰り返します。 注：希望の細菌希釈タイプと条件細菌のと同様に、正確な実験条件に依存します。例えば、10 -6 - 6.5ミリリットルNGT-3Dのチューブ当たり200細菌コロニー- OP50株大腸菌の10 -8希釈は1から生成するのに十分でした。 10 -7 -コロニーの数は60を超えていたとき、ワームは、10 -6の希釈で発生し、確実に開発され、正常に再生しました。 NGB-3Dの場合は、10 -8の希釈を使用します。 </li></ol> 3.メイキングNGT-3DとNGB-3D（200ミリリットル） <ol><li>細菌培養物を準備した後、0.6グラムのNaCl、1グラムの造粒寒天、および500mlのフラスコ内の0.5グラムペプトンを混ぜます。フラスコに磁気攪拌棒を挿入します。 </li><li> 195ミリリットルに蒸留水を加え、アルミホイルでフラスコの口をカバーしています。 </li><li> 15分間121℃でオートクレーブ。 </li><li>撹拌プレート上にホットオートクレーブ処理フラスコを置き、少なくとも2時間、中程度の速度で攪拌します。 40℃にフラスコを冷却します。完成した寒天の曇りにつながる可能性があり、ここで、高温でから継続して十分に冷却するようにしてください。しかしながら、より低い温度は、寒天の早期硬化をもたらすことができます。 <ol><li>撹拌プレート上に配置する前に、40℃の水浴で15分間でフラスコを置き、冷却プロセスをスピードアップする（オプション）。 </li></ol></li><li>寒天培地の温度が40℃に達したとき、溶液を攪拌し続けるように、200μlの1 MのCaCl 2、5 mg / mlでのコレステロール溶液200μl、200μlの1 MのMgSO 4および5 mlの1 M KPO 4緩衝液を加えます1mMのCaCl 2を、5μg/ mlのコレステロール、1mMのMgSO 4を、1 mMのKPO 4の最終濃度まで。 <ol><li> NGT-3Dの寿命アッセイのために（オプション）120μM12の最終濃度に150mMのFUDRの80μlを添加します。 </li></ol></li><li>直接フラスコにステップ2.4からの細菌培養物を希釈した10ミリリットルの6ミリリットルを追加します。 <ol><li> （オプション）NGT-3Dの場合は、ステップ3.6前の寒天培地の6ミリリットルを削除し、別にそれを暖かく保ちます。このメディアが使用されます細菌フリー上層。 </li></ol></li><li>滅菌手順を使用して、無菌培養室にメディアを分配します。チャンバーのカバーがしっかりと閉じていることを確認します。 <ol><li>ステップ3.7からのメディアが完全に硬化する前に、上のステップ3.6.1から細菌を含まない寒天培地の層を作る、寒天の上面に形成されるの細菌コロニーを防止するために、（オプション）。これはNGT-3Dの上部にある細菌フリーゾーンを作成します。 </li><li> NGT-3Dの場合は、細菌の寒天層を作り、そして慎重に薄いbacteria-を作るために半硬化3Dメディアの上に細菌を含まない培地200μlのを分配するために8ミリリットル透明なプラスチック製試験管に6.5ミリリットルのメディアを注ぎます上部の自由層。 </li><li> NGB-3Dの場合は、25cm 2の透明なプラスチックの細胞培養瓶にメディアの65ミリリットルを注ぎます。この量は、25cm 2の細胞培養ボトルのボディを埋める必要があります。 </li></ol></li><li>細菌コロニーが成長できるようにするために、室温で1週間垂直室を残します少なくとも直径1mmのかなりの大きさ。 <ol><li> （オプション）NGT-3Dで寿命アッセイのために、FUDRの光劣化を防止するために、アルミホイルでチャンバーをカバーしています。 </li></ol></li></ol> NGT-3D（相対同腹サイズアッセイ）上でワームの人口の4メジャーフィットネス<ol><li>ピックや細菌フリーNGMプレート上の転送白金線を使用して、L4段ワームを選びます。ワームは自由に体に付着した細菌を除去するために、数分間に移動できるようにします。 </li><li>ステップ4.1を繰り返して、このワームは細菌から自由であることを確認してください。一般的に、4.1の2反復が十分にあります。 </li><li>慎重に白金線ピックで3Dメディアの表面にきれいなワームを置きます。ワームは、最終的には3D寒天マトリックス中に寒天を入力する必要があります。 </li><li>ゆるくいくつかの空気がチューブに入るためにできるようにカバーを閉じますが、寒天培地の乾燥を防止します。 </li><li> 20℃で96時間インキュベートします。 </li><li> 4日後、しっかりと蓋を閉じて置きます88℃の水浴にNGT-3D培養チャンバは、寒天を融解します。熱は虫を殺すが、自分の体はそのまま残ります。 注：NGT-3Dのための8ミリリットルのチューブを使用して、20から30分間のインキュベーションは、通常は十分です。 </li><li>ガラスピペットを使用して9センチプラスチックシャーレ上に溶融したメディアを転送します。ワームは、多くの場合、それらに固執することができますようにプラスチックピペットは、お勧めされていません。 </li><li>送信立体解剖顕微鏡を用いて、L3、L4でワーム、および成体段階の数を数えます。 F1及びF2世代はここで混乱することができますように、L2ステージと若いワー​​ムをカウントしないでください。したがって、このアッセイは、相対的なひなサイズ分析ではなく、合計ひなサイズのアッセイです。 </li></ol> 5.イメージとNGB-3Dでの録音ワームの動作<ol><li>白金線を使用して、L4段ワームをピックし、細菌フリーNGMプレートに移し、それが自由に数分間移動させることにより、表面に付着した細菌を除去します。 </li><li>インクルードをめざし確実にする手順を繰り返し5.1rmが細菌から自由です。 </li><li>慎重に白金線ピックと瓶の首に近いNGB-3Dの寒天表面の中央にきれいなワームを配置します。ワームは、最終的には3D寒天マトリックス中に寒天を入力する必要があります。 </li><li>寒天培地の乾燥を防止しながら、緩やかにいくつかの空気がチューブに入るためにできるようにカバーを閉じます。 </li><li>画像または伝送ステレオ解剖顕微鏡下でワームを記録。ワームが3Dマトリックスを通って移動するようにフォーカスを調整します。 </li></ol>

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The authors have nothing to disclose.

古典的な線虫の増殖培地プレートを使用して、 線虫の実験室での栽培は、 線虫の研究が提供していることが重要な発見の数百に非常に重要でした。ここで、我々はより正確に自然な立体生息環境を反映している環境で、 線虫を育成する新たな方法を提示します。他の方法は、3D 13の線虫を観察するために使用されているが、これは、固体三次元マトリック...

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の建設は、寒天（ 図1A）全体に間隔をあけ小さな細菌コロニーを寒天で満たされた試験管になり、シンプルで簡単なプロトコルです。ワームは自由に細菌コロニーを発見し、消費し、寒天マトリックスを通って移動することができます。 C.エレガンスが再現し、NGT-3Dで正常に成長できるかどうかを確認するために、我々は標準的な2D NGMプレー?...

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Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

我々はNGT-3DとNGB-3Dと呼ばれる3D線虫の栽培システムを構築するための簡単な方法を提示します。これらは、標準的な2D実験室のC.エレガンス培養プレートより自然な線虫（Caenorhabditis elegans）の生息地に類似している生息地における線虫のフィットネスや行動を研究するために使用することができます。

の栽培<em&gt;線虫（Caenorhabditis elegans）</em&gt;ラボにおける三次元で

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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Biology

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

17.8K ビュー.  Yonsei University. 我々はNGT-3DとNGB-3Dと呼ばれる3D線虫の栽培システムを構築するための簡単な方法を提示します。これらは、標準的な2D実験室のC.エレガンス培養プレートより自然な線虫（Caenorhabditis elegans）の生息地に類似している生息地における線虫のフィットネスや行動を研究するために使用することができます。 

ビデオ: 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.

線虫の宿主微生物間相互作用を研究する多彩なIn Vivoモデル

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

遺伝学

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.

線虫の大規模栽培のプレート ・ ベース: 糖尿病の代謝変化に関する研究のための試料作製

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顕微鏡:ノマルスキ顕微鏡を用いた カエノラブディティス・エレガンス 胚発生の解明

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

発生生物学

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.

基本的な<em&gt;線虫Caenorhabditis elegans</em&gt;方法：同期との観測

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.

カエノラブディティス・エレガンスの長期増殖とイメージングのためのシンプルなマイクロ流体チップ

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

生物工学

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.

培養胚のための方法<em&gt; C.虫</em&gt;細胞

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.

培養<em&gt;線虫（Caenorhabditis elegans）</em &gt;微小粒子衝撃による無菌液体培地およびトランスジェニックワームの作成に

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.

カエモルハブ炎エレガンスにおける細胞構造と器官形態学の研究に関する定量的アプローチ

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.

縦断的蛍光モニタリングと嫌悪的介入のための固体培地上での個々の カエノラブディティスエレガンスの 長期培養

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.

マルチウェルデバイスの固体培地上での単離 されたカエノラブディティスエレガンスの 長期培養とモニタリング

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

の栽培

要約

さらに動画を探す

この動画の章

線虫の宿主微生物間相互作用を研究する多彩なIn Vivoモデル

線虫の大規模栽培のプレート・ベース: 糖尿病の代謝変化に関する研究のための試料作製

4D顕微鏡:ノマルスキ顕微鏡を用いたカエノラブディティス・エレガンス胚発生の解明

基本的な

カエノラブディティス・エレガンスの長期増殖とイメージングのためのシンプルなマイクロ流体チップ

培養胚のための方法

培養

カエモルハブ炎エレガンスにおける細胞構造と器官形態学の研究に関する定量的アプローチ

縦断的蛍光モニタリングと嫌悪的介入のための固体培地上での個々のカエノラブディティスエレガンスの長期培養

マルチウェルデバイスの固体培地上での単離されたカエノラブディティスエレガンスの長期培養とモニタリング

の栽培

要約

さらに動画を探す

この動画の章

線虫の宿主微生物間相互作用を研究する多彩なIn Vivoモデル

線虫の大規模栽培のプレート ・ ベース: 糖尿病の代謝変化に関する研究のための試料作製

4D顕微鏡:ノマルスキ顕微鏡を用いた カエノラブディティス・エレガンス 胚発生の解明

基本的な

カエノラブディティス・エレガンスの長期増殖とイメージングのためのシンプルなマイクロ流体チップ

培養胚のための方法

培養

カエモルハブ炎エレガンスにおける細胞構造と器官形態学の研究に関する定量的アプローチ

縦断的蛍光モニタリングと嫌悪的介入のための固体培地上での個々の カエノラブディティスエレガンスの 長期培養

マルチウェルデバイスの固体培地上での単離 されたカエノラブディティスエレガンスの 長期培養とモニタリング

線虫の大規模栽培のプレート・ベース: 糖尿病の代謝変化に関する研究のための試料作製

4D顕微鏡:ノマルスキ顕微鏡を用いたカエノラブディティス・エレガンス胚発生の解明

縦断的蛍光モニタリングと嫌悪的介入のための固体培地上での個々のカエノラブディティスエレガンスの長期培養

マルチウェルデバイスの固体培地上での単離されたカエノラブディティスエレガンスの長期培養とモニタリング