我々はS.タネジャ-バゲスワル、K. Beifuss、S. Kedroske、そしてHCに感謝したい。有用な議論のためのシャオ。この作品は、分子細胞医学のTAMHSC省からの資金投​​入を開始することによって資金を供給された。化合物の範囲や回転するディスクは、部門とディーンのTAMHSC Officeが提供する資金で購入した。一部の菌株は、研究資源のためのナショナルセンターによって資金を供給される線虫遺伝学センターによって提供されていました。 pRF4は（ROL - 6（su1006））A.火災の贈り物だった。

1。 DIIの調製染色<ol><li> DMF中20 mg / mLのDII（Biotium、（株）、ヘイワード、カリフォルニア州）のストック溶液を調製。 DIIは、敏感な光なので、ホイルで包むことによって光からDIIを保護する。 </li><li>各集団に対して399.4μLM9は0.6μLDIIの株式を追加することにより、DIIの希釈率を作成します。これは、30μgの/ M9のmLのDIIの最終的な希釈率を与える必要があります。これは、同時に複数の集団を染色するためにスケールアップすることができます。ホイルにチューブ（s）をラップすることによって、光からシールドDII。 </li></ol> 2。線虫の調製<ol><li>汚染されていない線虫の人口を含む60 mmのプレートを使用してください。静かにすべての幼生と成体の動物を緩めるために板の表面全体に円を描くように液体を渦巻くことでM9バッファーに0.5％トリトンの解X - 100を用いてプレートから動物を洗う。滅菌済み1.5 mLチューブに洗って移す。 </li><li>すぐに30秒間2000rpmで動物をスピンダウン。できるだけ多く渉取り外して廃棄チューブの底に動物の質量を邪魔することなく可能な限りernatant。 </li><li>残留トリトンX - 100を削減するために、M9バッファー、スピンを使って動物をすすぎ、そして上清を除去します。一度この手順を繰り返します。 </li><li>溶液中で動物を懸濁するためにチューブとボルテックスで短時間にM9の作業DIIの溶液400μLを加える。 </li><li> 20℃水平に光で保護された環境で350 rpmで3時間チューブを振る。必要に応じて、動物は染色のために16時間までインキュベートすることができる。 </li><li>バインドされていない染料の量を減らすために、20秒間2000rpmで動物をスピンダウン。削除して、動物の質量を乱すことなく、上清をできるだけ捨てる。 </li><li> 400μLM9バッファーで再懸濁し、動物とOP50 E.を接種したNGM寒天プレートの無菌の部分に水などの液体を注ぐ大腸菌 。動物は少なくとも30分を回復することができます。回復時間の間、動物は、DIIの染色液から、食品へはい出るてください。このステップフリーDIIからバックグラウンド蛍光が減少します。 </li></ol> 3。マウントと観察標本<ol><li>オートクレーブやマイクロ波を用いた水の4％の寒天を溶かす。 </li><li>スライドガラス上にラボのテープのレイヤーの2つの部分で、寒天のパッドの均一な厚さを確保するために使用できる再利用可能なスペーサーを、作成します。 twoスペーサ​​ースライドは合計してください。 </li><li> twoスペーサ​​ースライド間のきれいなガラスのスライドを手配する。きれいなスライドガラスの中央上に溶融4％寒天のピペット約150μL（4滴）。すぐに寒天のパッドを形成するために追加のスライドを使用して溶融寒天をカバーしています。慎重に取り付けスライドの上部を中心にパッドを保ち、カバースライドを削除します。 </li><li>パッドの上に - 線虫麻酔薬（1mMのレバミゾール、例えば100μM）の5μL約ピペット。 </li><li>マウント8 - 麻酔薬および顕微鏡のカバーガラスとカバーで12匹。 </li><li>少なくとも40倍objectiを装備した化合物または共焦点顕微鏡を用いて動物を観察<html> VEとTRITC / DsRedの（または他の互換性のある）フィルタ。 DIIの蛍光励起極大が549 nmであり、その排出量の最大値は、バインドされた色素（Biotium、（株）、ヘイワード、カリフォルニア州）のための565 nmである。 </li></ol> 4。代表的な結果野生型と変異体CのDIIの汚れは、クチクラエレガンス 。クチクラ表面はannulusの複数形、いくつかの段階では、円周畝で区切られて含まれ、長手方向の尾根はalaeと呼ばれる。それぞれの発達段階は、異なる組成2のクチクラ構造を持っています。 alaeとannulusの複数形の両方の尾根や畝は蛍光幼虫と大人の段階を通じて、表面組成に応じて、染色とまでこの方法を使用して回復の翌日から見えます。バックグラウンド蛍光斑点が、時には（図1、図2A - E、H、I）（図2F、G）観察ではなく、日常的にされています。すべての画像は、記載され、回転するディスク共焦点または、、広視野（WF）化合物の顕微鏡で撮影されました。 ENT&quot;&gt; <img alt="図1" src="/files/ftp_upload/3362/3362fig1.jpg" /> 図1。 DIIは、汚れキューティクルと環境にさらされるニューロンを 。 L2段の動物。 630x倍率。モザイク画像の部品は、iVision - Macのソフトウェア（バイオビジョンテクノロジーズ、エクストン、ペンシルバニア州）を用いて捕獲された。画像は、Adobe Photoshop CS3（アドビシステムズ社、サンノゼ、CA）を使用して結合した。スケールバー=10μmである。 DIIはまた、蛍光染色をそれぞれ頭と尾のamphidとナナフシ類の感覚ニューロン（矢印があるマークを付ける）。 <img alt="図2" src="/files/ftp_upload/3362/3362fig2.jpg" /> 図2。 DII蛍光染色C.後胚発生のすべての段階で虫がキューティクル 。 630x倍率。スケールバー=10μmである。 B）L2; C）dauer; D）L3; E）L4;）L1における野生型動物の染色およびF）大人のステージが。 alaeと環状の尾根は、蛍光（A、C）L1とdauer動物で染色される。 L2の動物のDIIの汚れ環状隆起部（B）。環状は、L3とL4の動物（D、E）で染色畝。 alaeの畝とannulusの複数形は、成熟した動物（FH）で染色されています。 Alaeは、動物の長さを実行する2つ、五、三尾根（L1、dauer、または成体動物で、それぞれ）（矢）16で構成されています。 annulusの複数形は、動物の周りの周方向の隆起（矢頭、FとJ）を作成します。成人変異動物のキューティクルは、中程度のクチクラ組織の欠陥（GH）を表示。 G）alaeの尾根は、（WF）不連続です。 H）リナalaeの尾根が融合され、分岐または（WF）二股されています。コラーゲン遺伝子の変異体はalaeと環状の組織の欠陥（IJ）を示す。 I）トランスジェニック動物において過剰発現しpRF4（ROL - 6（su1006））、動物の長さに斜めにalaeの嘘の尾根。トランスジェニック動物を過剰発現pRF4（ROL - 6（su1006））におけるJ）annulusの複数形が不規則なパターンが表示されます。 <img alt="図3" src="/files/ftp_upload/3362/3362fig3.jpg" /> 図3。 externをらの形態学的構造は、DIIの染色により照明されています。630x倍率は。スケールバーは=10μmである。 DIIも）成人雌雄同体外陰部、B）成人男性の尾線とファン、およびC）両性テールスパイク（この変異体バックグラウンドでforkされた）を含む他の外部の機能を、強調表示されます。 <img alt="図4" src="/files/ftp_upload/3362/3362fig4.jpg" /> 図4。キューティクルは、環境にさらされるニューロンよりも、DIIで染色するのに時間がかかります。 630x倍率。スケールバー=10μmである。染色の2時間後、amphid（図示せず）とナナフシ類の感覚ニューロン（矢印）十分に染色されています。対照的に、若い動物のキューティクルは、部分的にのみパッチ（矢頭）で染色される。 <table border="1"><tbody><tr><td> 洗う </td><td> 染色液（+ DII） </td><td> インキュベーション時間 </td><td> キューティクルのステンドグラス </td></tr><tr><td> M9 + 0.5％Trのiton X - 100 </td><td> M9 + 0.5％トリトンX - 100 </td><td> 2時間</td><td>ない</td></tr><tr><td> M9 + 0.5％トリトンX - 100 </td><td> M9 + 0.5％トリトンX - 100 </td><td> 3時間</td><td>ない</td></tr><tr><td> M9 + 0.5％トリトンX - 100 </td><td> M9 </td><td> 2時間</td><td>部分</td></tr><tr><td> M9 + 0.5％トリトンX - 100 </td><td> M9 </td><td> 3時間</td><td>はい</td></tr><tr><td> M9 + 0.5％トリトンX - 100 </td><td> H 2 0 </td><td> 2時間</td><td>部分</td></tr><tr><td> M9 + 0.5％トリトンX - 100 </td><td> H 2 0 </td><td> 3時間</td><td>はい</td></tr></tbody></table> 表1。異なる条件下でのクチクラ染色。様々なインキュベーションのソリューションおよび時刻は、動物でクチクラ染色を最適化するためにテストされました。 H 2 0、滅菌蒸留水。大人キューティクル汚れCも部分的な染色では、幼虫のクチクラの斑状の染色（図4）を示していますonsistently。

<ol>
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我々は、開示することは何もない。

ここで紹介するDIIの染色法は、C. elegansのキューティクルを視覚化するため、比較的迅速かつ便利な方法が可能になります。一般的に画像環境にさらされる感覚ニューロン15,17に使用される方法を再利用して最適化することにより、DIIは、蛍光alaeと環状構造（図1および図2）だけでなく、外陰部、男性の尾、および両性テールスパイクの両方を染色するために使用することができます（図3）。我々は、インキュベーション溶液と時間が常にキューティクル（表1、図4）を染色するためにDIIの能力に影響を及ぼすことを見出した。 DIIの染色環境にさらされるニューロンのための方法は、トリトンX - 100 15とM9で2時間のインキュベーション時間を使用しています。界面活性剤とM9の初期洗浄には、トリトンX - 100は、クチクラ表面から汚れを除去する手助けをし、凝集から動物を防ぐことができます。しかし、同じバッファ内の染色液中での動物三時間インキュベートするキューティクル（表1）染色から色素を防ぎます。これは、洗剤の溶液中で長期の治療によって取り除かれている線虫の表面の脂質が原因である可能性があります。 M9塩溶液はキューティクルのDIIの染色に必要とされていないことを示すDIIの汚れクチクラと水の動物のインキュベーション、。水ベースのプロトコルは神経DIIの染色15に推奨されていますが、我々は、動物がこの方法は頻繁に不健康な出現扱うことがわかります。 0.5％簡単にトリトンX - 100 M9での動物を洗濯、M9で作ったソリューションを染色で三時間のインキュベーションに続いて、クチクラ構造の一貫性のある染色を提供し、動物の健康を維持します。 動物を観察した後、救助され、直接ダウンストリームの解析を可能にして、維持することができます。このメソッドは、を含む、多くの研究で使用することができる便利なツールですが、クチクラ分泌および組織、表皮細胞の開発、異時性遺伝子経路、および線虫の進化に限定されない。

<table border="1"><tbody><tr><td> 試薬 </td><td> シノニム </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> DII </td><td> 1,1&#39; - ジオクタデシル- 3、3,3&#39;、3&#39; - tetramethylindocarbocyanine過塩素酸塩</td><td> Biotium、（株） </td><td> 60010 </td><td>株式の希釈： DMF中20 mg / mLの 希釈率：30 mg / mLの</td></tr><tr><td> DMF </td><td>ジメチルホルムアミド</td><td> Sigma - Aldrich社、（株） </td><td> D4551 </td><td></td></tr><tr><td>トリトン X - 100 </td><td>オクチルフェノキシポリエトキシエタノール</td><td> VWRインターナショナル、LLC。 </td><td> EM - 9410 </td><td></td></tr><tr><td> M9 </td><td> 22mmのKH 2 PO 4、42mmののNa 2 HPO 4、86mMのNaCl、1mMのMgSO 4を </td><td></td><td></td><td></td></tr><tr><td> NGM &lt;/ TD&gt; <td>線虫の増殖培地</td><td> IPMサイエンティフィック株式会社</td><td> 11006-501 </td><td> NGM寒天プロトコル18に従って調製することができる</td></tr><tr><td>寒天</td><td></td><td> EMDケミカルズ（株） </td><td> 1.01614 </td><td>水、4％ </td></tr><tr><td>レバミゾール</td><td>塩酸レバミゾール</td><td> Sigma - Aldrich社、（株） </td><td> 31742 </td><td> 100μM - 必要に応じて、1 mMのレバミゾール</td></tr><tr><td>顕微鏡スライド</td><td></td><td> VWRインターナショナル、LLC。 </td><td> 16005-106 </td><td></td></tr><tr><td>顕微鏡のカバーガラス</td><td></td><td> VWRインターナショナル、LLC。 </td><td> 16004-302 </td><td></td></tr><tr><td>化合物の範囲</td><td></td><td>カールツァイス株式会社</td><td> A1m </td><td>実験のニーズに合わせて目標を使用</td></tr><tr><td> TRITCまたは他の互換性のあるフィルタ</td><td> ＆ニッポン放送p; </td><td>クロマテクノロジー株式会社</td><td> 49005 </td><td> ET - DsRedタンパク質は、（TRITC/Cy3）フィルタセットをスパッタ</td></tr></tbody></table>

visualization of caenorhabditis elegans cuticular structures using the lipophilic vital dye dii

C.のクチクラelegansは動物1-4の外側を取り囲む非常に強い構造です。キューティクルだけではなく環境から動物を保護するだけでなく、ボディの形状を決定し、運動性4-6に役割を果たしている。表皮細胞から分泌されるいくつかの層は最も外側の脂質層7を含む、キューティクルを構成しています。 キューティクルの円周尾根はannulusの複数形のパターン動物の長さと呼ばれ、開発8のすべての段階で存在している。 AlaeはL1、dauer、そして大人のステージ2,9を含む開発の特定の段階で、中に存在する縦方向の尾根です。クチクラコラーゲンの組織に影響を及ぼす遺伝子の変異は、クチクラ構造と動物の体の形態5,6,10,11を変更することができます。 DIC光学系と化合物の顕微鏡を使用してクチクラのイメージングが可能ですが、クチクラ構造を強調する現在の方法は、蛍光を含むセントジーン発現12、抗体染色13、および電子顕微鏡1。ラベルの付いた小麦胚芽レクチン（WGA）もクチクラ糖タンパク質を可視化するために使用されていますが、細かいクチクラ構造14を解決するために制限されています。蛍光色素を使用してクチクラ表面の染色が観察されたが、詳細に15に特徴がないされています。我々は、ライブCでキューティクルを可視化する手法を提案一般的にC言語で使用されている赤色蛍光親油性色素DII（1,1&#39; -ジオクタデシル- 3、3,3&#39;、3&#39; - tetramethylindocarbocyanine過塩素酸塩）を、使用して虫elegansの環境にさらされるニューロンを可視化する。 DIIの染色のためのこの最適化されたプロトコルは、annulusの複数形、alae、外陰部、男性の尾、およびCの雌雄同体尾のスパイクの高分解能蛍光可視化するための、シンプルで堅牢な方法です。 エレガンス 。

Watch this Scientific Journal Video about Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI at JoVE.com

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

我々は、ライブでキューティクルを可視化する手法を提案<em&gt; C.エレガンス</em&gt;一般的に使用される赤色蛍光親油性色素DII（1,1&#39; - ジオクタデシル- 3、3,3&#39;、3&#39; - tetramethylindocarbocyanine過塩素酸塩）を使用して、<em&gt; C.エレガンス</em&gt;環境的に露出した神経細胞を可視化する。この最適化されたプロトコルで、alaeと環状のクチクラ構造は、DIIによって染色し、化合物の顕微鏡を用いて観察される。

の可視化線虫（Caenorhabditis elegans）</em親油性生体染色色素、DIIを使用する>キューティクルの構造

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

visualization-caenorhabditis-elegans-cuticular-structures-using

Nanyang Technological University

Research

JoVE Journal

Biology

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

17.0K ビュー.  Texas A&M University System Health Science Center, College of Medicine. 我々は、ライブでキューティクルを可視化する手法を提案 C.エレガンス一般的に使用される赤色蛍光親油性色素DII（1,1' - ジオクタデシル- 3、3,3'、3' - tetramethylindocarbocyanine過塩素酸塩）を使用して、 C.エレガンス環境的に露出した神経細胞を可視化する。この最適化されたプロトコルで、alaeと環状のクチクラ構造は、DIIによ...

ビデオ: Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

計測線虫（Caenorhabditis elegans）</em96穴マイクロタイタープレートの>寿命

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

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 ...

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

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

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

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

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

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

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 ...

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

血管視覚化のためのDiI灌流<em&gt; Ambystoma mexicanum</em

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

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 ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

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

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

細胞外小胞体のカエノールハブディティス・エレガンスの精製と分析

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

カエノハブディティス・エレガンスを使用して組織特異的シャペロン相互作用をスクリーニングする

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

Caenorhabditis elegansにおける凝集定量の自動化

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

カエノラブディティス・エレガンスゲノムDNAの小規模抽出

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and ...

の可視化線虫（Caenorhabditis elegans）キューティクルの構造