我々は、サミュエルウォードに感謝、ダイアンC.はこの手法を開拓するためシェイクスとグレゴリーネルソン。我々は、活性化精子を示すビデオをパヴァーヌKadandaleとブライアンGeldzilerに感謝、有用な議論のためのシンソンの研究室のメンバーは、CGC株とNIHの助成金（R01HD054681）を通じて私たちをサポートするために。

男性人口の1）濃縮<ol><li>実験的な必要性に応じて、男性の多数は、次のいずれかの方法を用いることにより得ることができます。 <ol><li>野生型の雄の大規模な人口は、NGMプレートの中央にOP50シードの小さな芝生の5野生型の男性と1雌雄同体を横断することによって得ることができる。後続世代の約50％が野生型の男性になります。 </li><li> 彼- 5（e1490）または彼- 8（e1489）雌雄同体は彼- 5。男性の多数を投げると彼- 8男性は肥沃であり、精子の形態と機能には明らかな欠陥はありません。そう、 彼- 5または彼- 8の男性は、多くの実験のために野生型の男性の代わりに使用することができます。 </li></ol></li></ol>男性の2）の同定と分離<ol><li>ワームの尾の形態を調べ、男性、ワームの尻尾は丸められます。 </li><li> L4ステージの男性を選んで、E.を接種したNGMプレートに移す大腸菌 OP50と、それらが一日か二日のために成長しましょう。雌雄同体の存在下で成長している独身男性は精子の損失を防ぐため、精子の数が多いが、実験手順の実行中に利用できるようになる。 </li><li>解剖の日に、細菌なしでNGMプレートに上の独身男性を転送し、それらを数分間クロールができます。このステップでは、E.の小さい層を除去するのに役立ちます大腸菌は、ワームの表面に付着。また、バッファに時計のガラスと転送雄ではM9バッファー（1リットル当たり6グラムのNa 2 HPO 4、3G KH 2 PO 4、5グラムのNaCl、0.25グラムのMgSO 4 · 7H 2 O）のピペット少量。 </li></ol> 3）男性とin vitroでの活性化の解剖は、 <ol><li>スライドガラスを取り出し、PAPのペンを使って小さな円をマーク。ピペット1X精子中期の30〜50μL（50mmのHEPES、25mMのKClを、45ミリのNaCl、1mMのMgSO 4を用い 、5mMの塩化カルシウム、10mMのブドウ糖、pH7.8）中の円内。疎水性の円は、その境界内の精子の培地を維持するのに役立ちます。 </li><li>精子の培地に5-10人の男性を転送します。 </li><li>顕微鏡下でスライドを表示して、あなたの手の両方で27ゲージの針を取り付けた2つのシリンジを保持する。 </li><li>ワームを&quot;ホールド&quot;と精子を放出する男性の後端をカットするために他の針を使用するもの針を使用してください。 </li><li>ワームをカットすると、通常は瞬時に精子細胞の膨大な数を排出。精子細胞は、実体顕微鏡下で分&quot;顆粒&quot;として表示されます。しかし、時には一部または精子細胞のほとんどは、死体の中に保持されることがあります。その場合には、静かにスライドガラス上の死体&quot;をドラッグすると、&quot;死体からの精子の放出を容易にすることができる。 </li><li>精子細胞を有効にするには、プロナーゼE（20μg/ml）を含む1X精子のミディアムでワームを細かく分析し、それらを5分間放置します。 </li><li>スライドは、実験の任意の時点でドライになるようにしてください。必要に応じてより多くの1X精子のミディアムを追加。 </li></ol> 4）spermtidsと精子の可視化<ol><li>優しく解剖試料の表面にカバースリップを配置。 </li><li>低倍率（10倍）下で精子のフィールドを見つける</li><li>通常100X油浸で - 精子または精子の細部がより高い倍率に移動して観察することができます。 </li></ol> 5）代表者の結果男性の線虫から単離された精子のDIC画像の例を図1に示されています。精子は球形で、核が顕著である。 試験管内活性化の精子では 、図2に示す。 <img src="/files/ftp_upload/2336/2336fig1.jpg" alt="図1" /> 図1℃のDIC画像線虫の精子。 <img src="/files/ftp_upload/2336/2336fig2.jpg" alt="図2" /> 図2。 試験管内活性化C言語でのDIC画像elegansは精子。

<ol>
	<li>Kimble, J., Crittenden, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controls+of+germline+stem+cells,+entry+into+meiosis,+and+the+Sperm/Oocyte+decision+in+Caenorhabditis+elegans.">Controls of germline stem cells, entry into meiosis, and the Sperm/Oocyte decision in Caenorhabditis elegans.</a> Annual Review of Cell and Developmental Biology. 23, 405-433 (2007).</li><li>Lhernault, S. W., Roberts, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Cell Biology. , 273-301 (1995).</li><li>Hodgkin, J., Horvitz, H., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nondisjunction+Mutants+of+the+Nematode+Caenorhabditis+elegans.">Nondisjunction Mutants of the Nematode Caenorhabditis elegans.</a> Genetics. 91, 67-94 (1979).</li><li>Altun, Z. F., Hall, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+male+C.+elegans+anatomy.">Introduction to male C. elegans anatomy.</a> WormAtlas. , (2008).</li><li>Shakes, D., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+spermiogenesis+in+C.+elegans:+a+pharmacological+and+genetic+analysis.">Initiation of spermiogenesis in C. elegans: a pharmacological and genetic analysis.</a> Dev Biol. 134, 189-200 (1989).</li><li>Nelson, G. A., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle+fusion,+pseudopod+extension+and+amoeboid+motility+are+induced+in+nematode+spermatids+by+the+ionophore+monensin.">Vesicle fusion, pseudopod extension and amoeboid motility are induced in nematode spermatids by the ionophore monensin.</a> Cell. 19, 457-464 (1980).</li><li>L'Hernault, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+and+cell+biology+of+spermatogenesis+in+the+nematode+C.+elegans.">The genetics and cell biology of spermatogenesis in the nematode C. elegans.</a> Molecular and Cellular Endocrinology. 306, 59-65 (2009).</li><li>Chatterjee, I., Richmond, A., Putiri, E., Shakes, D., Singson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+spe-38+gene+encodes+a+novel+four-pass+integral+membrane+protein+required+for+sperm+function+at+fertilization.">The Caenorhabditis elegans spe-38 gene encodes a novel four-pass integral membrane protein required for sperm function at fertilization.</a> Development. 132, 2795-2808 (2005).</li><li>Xu, X., Sternberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+C.+elegans+sperm+TRP+protein+required+for+sperm-egg+interactions+during+fertilization.">A C. elegans sperm TRP protein required for sperm-egg interactions during fertilization.</a> Cell. 114, 285-297 (2003).</li><li>Nelson, G., Roberts, T., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+spermatozoan+locomotion:+amoeboid+movement+with+almost+no+actin.">Caenorhabditis elegans spermatozoan locomotion: amoeboid movement with almost no actin.</a> J Cell Biol. 92, 121-131 (1982).</li><li>Machaca, K., DeFelice, L. J., L'Hernault, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+chloride+channel+localizes+to+Caenorhabditis+elegans+spermatids+and+chloride+channel+blockers+induce+spermatid+differentiation.">A novel chloride channel localizes to Caenorhabditis elegans spermatids and chloride channel blockers induce spermatid differentiation.</a> Dev Biol. 176, 1-16 (1996).</li><li>LaMunyon, C., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+viability+of+mutant+and+manipulated+sperm+by+artificial+insemination+of+Caenorhabditis+elegans.">Assessing the viability of mutant and manipulated sperm by artificial insemination of Caenorhabditis elegans.</a> Genetics. 138, 689-692 (1994).</li></ol>

利害の衝突は宣言されません。

SPE変異体を分析することに加えて、このプロトコルは、加齢に伴う精子の形態を分析するなど、他の重要なアプリケーションを、持っています。このプロトコルを使用して単離した精子と精子は野生型と変異体精子8、9、スライド10、生理学的測定9、11、あるいは人工授精12日の精子の運動性の免疫染色、などの他の下流の実験に使用することができます。

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<td>Tuberculin syringe</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>309623</td>
<td>Syringe: 1 ml, needle: 27G X &frac12; in</td>
</tr>
<tr>
<td>Pap pen</td>
<td>&nbsp;</td>
<td>Zymed</td>
<td>00-8888</td>
<td>&nbsp;</td>
<tr>
<td>VWR VistaVision HistoBond Microscope Slides</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>16004-406</td>
<td>Dimension: 75X25X1mm</td>
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<td>Cover glass</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>48366045</td>
<td>&nbsp;</td>
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<td>Protease</td>
<td>&nbsp;</td>
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isolation and in vitro activation of caenorhabditis elegans sperm

男性と雌雄同体の線虫 C. elegansの2つの自然に存在性的なフォームです。アメーバ精子は、男性と雌雄同体の両方で生産されています。約300 - - 配偶子形成の初期段階では、雌雄同体の生殖細胞は精子の数が限らへと分化し、貯精嚢と呼ばれる小さな&quot;袋&quot;に格納されています。その後、雌雄同体は、継続的に卵母細胞 1を生成する。対照的に、男性は自分の成人期を通じて独占的に精子生成。男性はそれが典型的な大人のワーム2の全細胞の&gt; 50％を占めていることそんなに精子を生成する。したがって、男性から精子を分離する雌雄同体のものと比べて簡単です。 男性のわずかな割合は、自然にX染色体3の自発的な非分離のために生成されます。より便利に、男性またはで雌雄同体を横断、彼を生じさせる変異の導入（男性の発生率が高い）表現型は1つが男性の人口3を豊かにできるような戦略の一部です。 男性は、簡単に尾の形態4を観察することにより、雌雄同体と区別することができる。雄の尾部が嵌合構造で丸められるのに対し、両性具有の尾は、指摘されている。 テールをカットすると、雄の生殖器官の内部に格納されて精子細胞の膨大な数を解放します。解剖は、27ゲージの針を使用して実体顕微鏡下で行われる。精子細胞が物理的に他のセルと接続されていないので、油圧は精子2を含む男性の体の内部の内容を、排出します。 男性は、直接&quot;精子の中&quot;の小滴に解剖されています。精子は、pHの変化に敏感です。したがって、HEPES、良好な緩衝能を持つ化合物は、精子のメディアで使用されています。グルコースと精子のメディアに存在する他の塩は、精子の整合性を維持するために浸透圧を維持するのに役立ちます。 精子への精子の後の減数分裂分化は、精子や精子の活性化と呼ばれます。 5揺れ、そしてネルソン6以前に円形精子細胞はここでは、Cのin vitroでの精子で実証プロナーゼE.を含む様々な活性化合物を添加することにより精子に分化誘導できることを示したプロナーゼEを使用して線虫の精子成功した精子は生殖能力の前提条件であり、したがって、精子形成の欠損変異株が無菌である。これまでいくつかの変異体は、特に精子形成の過程7で不良品であることが示されている。異常が小説SPE（精子形成の不良）のin vitroでの活性化に変異体は、私たちはこのイベントに参加する追加のプレーヤーを知る手助けになる際に発見。

Watch this Scientific Journal Video about Isolation and In vitro Activation of Caenorhabditis elegans Sperm at JoVE.com

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

男性から精子を分離し、活性化するためのプロトコル<em&gt; C.エレガンス</em&gt;ここで説明されています。男性のリリースの精子細胞の後端をカット。精子細胞は、プロテアーゼの添加によって活性化することができます。

アイソレーションと試験管内で</emの>活性化線虫（Caenorhabditis elegans）精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

isolation-and-in-vitro-activation-of-caenorhabditis-elegans-sperm

Research

JoVE Journal

Biology

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

16.2K ビュー.  Rutgers University. 男性から精子を分離し、活性化するためのプロトコル C.エレガンスここで説明されています。男性のリリースの精子細胞の後端をカット。精子細胞は、プロテアーゼの添加によって活性化することができます。

ビデオ: Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

固体プレートベースの食餌制限線虫（Caenorhabditis elegans）</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

生物学

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

の初期発生のタイムラプス顕微鏡線虫（Caenorhabditis elegans）

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

から細胞外液のヴァンパイア·アイソレーション<em&gt;線虫（Caenorhabditis elegans）</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

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 ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

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

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

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

におけるプロスタグランジンの抽出と分析<em&gt;線虫（Caenorhabditis elegans）</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

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 ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

結合されたヌクレオチドおよび線虫のタンパク質抽出

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

そのまま線虫の酸素消費量の測定

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

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 ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

カエノハブディティス・エレガンスにおける年齢関連神経変性疾患のモデル化

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...

アイソレーションと試験管内で活性化線虫（Caenorhabditis elegans）精子