我々は、Cの線虫遺伝学センターを認めますエレガンス株 。資金援助は、テリー·フォックス·研究所から提供されました。また、RolandeとマルセルGosselin大学院学生の身分とマギル統合がん研究研修プログラムのがん研究FRN53888でCIHR / FRSQ訓練助成金からEPに付与されたサポートを認めます。

試薬の調製<ol><li> C.のためのメディアの準備エレガンスの成長（ここでは、野生型動物とflcn-1（ok975）変異動物）。 <ol><li>トリスHCl、トリス塩基、Bactopetoneの31グラム、塩化ナトリウムを20gとコレステロールの0.08グラムの2.4グラムの5.5グラムを含有する修飾Youngren唯一バクトペプトン（MYOB）ドライミックスを準備します。振とうしながらよく混ぜます。 注：この混合物は、MYOB培地10 Lを調製するのに十分です。 注：通常の増殖培地（NGM）プレートはMYOB板27の代わりに使用することができます。しかし、同じタイプのプレートは、系統間の独立した反復間有効な比較のために使用されるべきです。このプロトコルでは、我々は唯一のMYOBのプレートを使用していました。 </li><li> MYOB乾燥混合物の6グラム、寒天の17グラムを追加することで、MYOB媒体の1 Lを調製し、122℃で45分間、H 2 O、オートクレーブで1 Lに構成しています。プレートを注ぎ、室温で2日間乾燥させてください。 </li><li>無菌技術を用いて、cultu 100mlの接種E.とLBブロス培地の再大腸菌 OP50細菌と37℃でO / Nを成長させます。 </li><li> E.と種子MYOBプレート大腸菌 OP50細菌。それは48時間室温で成長できるようにします。 </li></ol></li><li> RNAiを用いて遺伝子のダウンレギュレーションのための準備<ol><li> MYOBのRNAi送りプレートを準備します。ステップ1.1.1と1.1.2のように進みます。オートクレーブ処理後、培地を約55℃まで冷却し、1ミリリットルM 1のイソプロピルβ-D-1-チオガラクトピラノシド（IPTG）および1ミリリットルの100mMアンピシリンを追加することができます。 </li><li>ストリークE.大腸菌 HT115制御EVまたはプレート寒天アンピシリン上flcn-1特異的RNAi（50μg/ mlの）/テトラサイクリン（15μgの/ ml）のいずれかを発現するプラスミドで形質転換された細菌。 37℃で培養O / Nを成長させます。 </li><li>コロニーを選択し、Eを接種制御EVまたはflcn-1特異的RNAiのいずれかを発現し、37＆＃でLB /アンピシリン（50μg/ ml）中、O / Nでの培養を成長させるプラスミドで形質転換した大腸菌 HT115細菌176; C。 </li><li> E.によるシード·プレート大腸菌 HT115 EV細菌やHT115 flcn-1のRNAi細菌。 注：使用前にRTでプレートを48時間にしてください。給電によるのRNAiノックダウンが有効になっている場合は、DS-RNA 28の送達の他の方法を使用します。 </li></ol></li><li>パラコート（PQ）を使用して酸化ストレスを誘導します。 <ol><li> 7 H 2 O·KH 2 PO 4、のNa 2 HPO 4、5gのNaClをし、MgSO 4 0.25gの6gの3gを混合することによってM9バッファーを調製します蒸留水で1リットルに起動します。 RTで122℃、店舗ボトルで45分間オートクレーブソリューション。 </li><li>アッセイの日に、M9 / PQを含む溶液を準備します。このプロトコルは、100 mMのPQを使用します（M9バッファーの4ミリリットルに溶解PQの0.1028グラムを、ウェル当たりM9 / PQの40μlの全96ウェルプレートを埋めます）。 注意：PQが危険と毒性の高い化合物です。適切なガイドラインに従って処理します。 注：最初に新しいのを実行しています電車や新たな条件、それは7-10時間以内に動物を殺すために使用するのに最適な濃度を決定するためにPQの濃度を増加さに生存をアッセイすることをお勧めします。 </li></ol></li></ol>年齢同期L4人口の調製<ol><li> E.を接種した新鮮なMYOBプレートに20妊娠大人の雌雄同体を転送大腸菌 OP50細菌。アッセイの間に必要な虫の数に基づいて、いくつかのプレートを準備します。 </li><li>彼らは6時間産卵することを可能にした後、白金線虫を用いて、母親を削除選びます。産卵数を評価するために顕微鏡下でプレートを確認してください。 </li><li>卵が孵化し、48時間、20℃で成長させます。この時点で、動物が後期L4 /若年成人の段階にあります。同じステージの動物を選択し、PQ液含有ウェルに移します。 注：48時間のインキュベーションが唯一の野生型動物と同様に成長する突然変異体に適用されるので、それが発達監視することが重要ですそれはそうでなければ、他のタイムラインを使用する必要があり、野生型の現像速度に準拠を保証するために、新たにテストされた変異体の割合。 </li><li>あるいは、同期技術​​を使用しています。次亜塩素酸溶液を用いて、L1幼虫ワームの同期人口を（;光から分離するためにアルミホイルで瓶を包む水25ml、5.25％の次亜塩素酸ナトリウムの125ミリリットル、4 M NaOHを50ミリリットル）を生成します。強烈な漂白がアッセイ中に動物の行動に影響を与える可能性があるので、このアッセイのために、それは、推奨されません。 注意：ガイドラインに従って次亜塩素酸溶液を処理します。 注：ダウン、目的の遺伝子を調節する特異的RNAi細菌を播種したRNAiプレートに母親の転送を除いて、セクション2で説明したように雌雄同体を同期させるためにRNAiを使用する場合。 RNAiのでノックダウンが弱い場合には、複数の世代のための給紙を推奨します。 </li></ol> 3.酸化ストレス抵抗性アッセイを&lt;実行/ P&gt; <ol><li> 96ウェルプレートの各ウェルに100 mMのPQ溶液をピペット40μL（新しく調製）。すべての条件（処理、突然変異体など ）を複製するように、少なくとも12のウェルを使用してください。白金線虫ピックを使用して、十分に開始時刻と終了時刻を示すあらゆるに5-8 L4幼虫動物を転送します。 </li><li>別の条件を開始すると、開始時刻と終了時刻を確認します。ワームを転送し、開始時刻から時間後に死亡した動物の得点間のインキュベーション時間で2​​0℃でプレートを置きます。 </li><li>解剖顕微鏡を使用して、生存時間ごとにカウント。ゆっくりカウントを開始する前に、プレートを振ります。死んだ動物として、あっても高強度の光をスポットした後、移動しないワームを示しています。また、生きている動物の数を示しています。 注：高倍率でワーム形状、尾と頭の動きを見ると、生きているから死んだ虫を決定するのに役立ちます。 </li><li>ほとんどのワームは死んでいるまで、この手順を時間ごとに繰り返します。 </li></ol> &lt;Pクラス= &quot;jove_title&quot;&gt; 4。毎回ポイントでの生存割合の決意<ol><li>すべてのウェルに移した動物の数を追加することによって、条件あたりの動物の数を計算します。破損または転送時に殺される動物を無視します。 </li><li>すべての時点について、条件ごとに死んだ動物の合計数（12ウェル中の死んだ動物の合計）を計算します。すべての時点について、死亡率（死亡した動物の数の動物の総数で割り、100を掛けた）及び生存率（100パーセントマイナス死）を計算します。 </li><li>このアッセイを、少なくとも3つの独立した回を繰り返します。 </li></ol>

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S., Curtis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+AMP-activated+protein+kinase+AAK-2+links+energy+levels+and+insulin-like+signals+to+lifespan+in+C.+elegans.">The AMP-activated protein kinase AAK-2 links energy levels and insulin-like signals to lifespan in C. elegans.</a> Genes Dev. 18, 3004-3009 (2004).</li><li>Lee, H., et al. <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+AMP-activated+protein+kinase+AAK-2+is+phosphorylated+by+LKB1+and+is+required+for+resistance+to+oxidative+stress+and+for+normal+motility+and+foraging+behavior.">The Caenorhabditis elegans AMP-activated protein kinase AAK-2 is phosphorylated by LKB1 and is required for resistance to oxidative stress and for normal motility and foraging behavior.</a> J Biol Chem. 283, 14988-14993 (2008).</li><li>Honda, Y., Honda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+daf-2+gene+network+for+longevity+regulates+oxidative+stress+resistance+and+Mn-superoxide+dismutase+gene+expression+in+Caenorhabditis+elegans.">The daf-2 gene network for longevity regulates oxidative stress resistance and Mn-superoxide dismutase gene expression in Caenorhabditis elegans.</a> FASEB J. 13, 1385-1393 (1999).</li><li>Restif, C., Metaxas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+swimming+motions+of+C.+elegans+worms+with+applications+in+aging+studies.">Tracking the swimming motions of C. elegans worms with applications in aging studies.</a> Med Image Comput Comput Assist Interv. 11, 35-42 (2008).</li><li>Buckingham, S. D., Sattelle, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast,+automated+measurement+of+nematode+swimming+(thrashing)+without+morphometry.">Fast, automated measurement of nematode swimming (thrashing) without morphometry.</a> BMC Neurosci. 10, 84 (2009).</li></ol>

The authors declare that they have no competing financial interests.

C.エレガンスは、それが容易に培養することができるので、 生体内での遺伝的に酸化ストレス耐性を研究するための魅力的なモデル生物であり、急速に遺伝的に同一の子孫の多くをもたらします。酸化ストレス抵抗性を測定するための複数の方法は、以前に記載されており、それらは、PQ、ロテノン、H 2 O 2、及びジュグロン25,26,29-32などの種々のROS光源?...

A reference was corrected in the publication of <a href="http://www.jove.com/video/52746">Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates</a>. There was an error with reference 34 in the Citations section. It has been updated from:
Allen, E., Walters, I.B., Hanahan, D. Brivanib a dual FGF/VEGF inhibitor,is active both first and second line againstmouse pancreatic neuroendocrine tumors developing adaptive/evasive resistance toVEGF inhibition. Clin Cancer Res. 17, (16) 5299-310 (2011).
to:
Allen, A. T., Maher, K. N., Wani, K. A., Betts, K. E., &#38; Chase, D. L. Coexpressed D1- and D2-like dopamine receptors antagonistically modulate acetylcholine release in Caenorhabditis elegans. Genetics. 188, 579-590 (2011).

Erratum: Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

真核生物では、ミトコンドリアの電子伝達系で起こる酸化的リン酸化は、ATPの形でエネルギー生産の主な要因です。活性酸素種（ROS）は、このプロセスの自然な副産物です。シグナル伝達分子としての重要な役割にもかかわらず、過剰なROSは、DNA損傷、タンパク質のカルボニル化、および脂質酸化につながることができます。 ROS産生と解毒の間の不均衡は、エネルギーの枯渇につながる酸化ストレス、細胞の損傷を引き起こし、細胞死1,2をトリガします。酸化ストレスは老化および癌、糖尿病、心血管および神経変性疾患3-9を含む多くの生命を脅かす疾患の発展に貢献しています。 細胞は適切なROSレベルを維持し、酸化的損傷1,2に対してその成分を保護するために酵素的および非酵素的防御戦略を進化させてきました。スーパーオキシドジスムターゼ（SOD）の酵素はconveに第一幕RTスーパーオキシド後カタラーゼまたはペルオキシダーゼ酵素によって水に変換されるH 2 O 2へ。非酵素的な防衛戦略は、主に必須の細胞成分を保護する細胞高分子と比較して、ROSと速く反応する分子を含みます。 ROS解毒酵素の保護の役割にもかかわらず、いくつかのROS分子は、抗酸化防御機構を脱出し、酸化的損傷につながります。検出、修復、および損傷した細胞成分の分解は、酸化ストレス1,2間に本質的な防御戦略です。 ストレス耐性、特に酸化ストレスに関与するシグナル伝達経路は、進化的に高度10,11に保存されています。生物の条件が部分的にしか再現されている細胞培養実験とは異なり、モデル生物における酸化ストレスの研究12,13は、大きな意義を持っている。C.エレガンスは、CUL、容易かつ安価にすることができます自由生活線虫であります寒天培地上の細菌叢上tured。それは、（長さ約1mm）小型で、通常の遺伝子操作を容易に自己受精ふたなり、として成長します。それは大規模な遺伝子スクリーニング14を実行するための強力なツール作り、世代あたり約300の子孫を生成する、迅速なライフサイクルと高い生殖能力を有しています。 C.エレガンスゲノムが完全に配列決定され、遺伝子の40〜50％は、ヒトの疾患関連遺伝子15〜18の相同体であると予測されます。 RNAiを用いて、目的の遺伝子のノックダウンは、迅速かつCで簡単です。 エレガンス。遺伝子のダウンレギュレーションはE.動物に供給することによって達成することができます関心19のmRNAを標的とする二本鎖RNAを発現するプラスミドを保有する大腸菌細菌。そのため、大規模なRNAiスクリーニングを使用して、遺伝子機能の決意は、癌20,21を含むヒトの疾患を理解する上で大きな影響を与えています。 Oの研究Cでxidativeストレス耐性エレガンスは、酸化ストレスに対する抵抗13,22の保存されたメカニズムの同定につながりました。同定されたいくつかの経路は、低酸素、熱、および浸透圧ストレスなど、他のストレスに長寿と抵抗を調節する一般的な経路です。これらの経路は、インスリンシグナル伝達、TORシグナリング、およびオートファジーを含みます。その他の主な経路は、そのような、または損傷修復におけるこのような熱ショックタンパク質とシャペロン11,13,22としてスーパーオキシドジスムターゼ酵素とカタラーゼ酵素としてROSの解毒を伴います。 このプロトコルは、Cの酸化ストレスに対する抵抗性を決定する方法について説明します液体中のエレガンス 。我々は以前Cでflcn-1（ok975）の喪失時の酸化ストレスに対する抵抗性の増加を示しているので、私たちはプロトコルを示すためにflcn-1（ok975）と野生型動物を使用しましたエレガンス 23。また、この増加した抵抗性が依存することを示していますAMPKとオートファジーは、細胞生体エネルギーを改善し、ストレス耐性23を促進するシグナル伝達軸上。 PQは、反応性酸素種24を生成するために電子伝達系を妨害する酸化的ストレッサーです。同じアッセイを適合させることができ、他のROS源またはROSの生成化合物は、H 2 O 2およびロテノンとして使用することができます。同様のアッセイは、PQの低濃度は25,26を使用しているプレート上で開発されています。このアッセイの利点は、非常に高速であり、結果は、一日で得られることがあります。さらに、96ウェルプレート中の酸化ストレス耐性アッセイを行うために使用される液体の総体積はPQ含有プレートを調製するために使用される量に比べて低いです。したがって、使用PQの量は、液体アッセイであり、安価なアッセイをレンダリングし、有害廃棄物の生産を制限する、低いです。しかし、プレートアッセイと比較して、このアッセイの制限はラを含みます液体アッセイにおける食品のCKと空気と比較して、液体中の酸​​素濃度が低いです。これらは、いくつかのケースでは、結果に影響を与える可能性がある重要な要因です。したがって、このアッセイで得られた結果を支持することが推奨される酸化ストレス耐性の他の方法を使用して再現性を確認しました。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Agar bacteriological grade</td>
			<td>Multicell</td>
			<td>800-010-LG</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological peptone</td>
			<td>Oxoid</td>
			<td>LP0037</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride biotechnology grade</td>
			<td>Bioshop</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>C8503-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure tris hydrochloride</td>
			<td>Invitrogen</td>
			<td>15506-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris aminomethane</td>
			<td>Bio Basic Canada Inc</td>
			<td>77-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-202185A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Bioshop</td>
			<td>69-52-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Bio Basic Inc.</td>
			<td>8013-01-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl viologen dichloride hydrate</td>
			<td>Aldrich chemistry</td>
			<td>856177-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 60x15mm</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet 10ml</td>
			<td>Fisher</td>
			<td>1367520</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>G-Biosciences</td>
			<td>RC-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma</td>
			<td>M-5921</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Bioshop</td>
			<td>7558-79-4</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Discovery v8 stereo zeiss microscope</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 well clear microtiter plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>flcn-1 RNAi source</td>
			<td colspan="2">Ahringer Library</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring oxidative stress resistance of caenorhabditis elegans in 96-well microtiter plates

活性酸素種の産生と解​​毒の間の不均衡の結果である酸化ストレスは、心血管および神経変性疾患、糖尿病、老化、および癌を含む慢性ヒトの疾患、の主要な原因です。したがって、細胞系においても全生物を使用していないだけで酸化ストレスを研究することが重要である。C.エレガンスは、高度に進化的に保存されている酸化ストレスシグナル伝達経路の遺伝学を研究するための魅力的なモデル生物です。 ここでは、Cの酸化ストレス耐性を測定するためのプロトコルを提供します液体中のエレガンス 。簡単に言えば、そのようなパラコート（PQ）およびH 2 O 2などのROS誘導試薬はM9緩衝液に溶解され、溶液は、96ウェルマイクロタイタープレートのウェルに等分されます。シンクロナイズドL4 /若年成人C.エレガンス動物をウェルに転送される（5~8匹/ウェル）、生存率が測定されますほとんどのワームは死んでいるまで、毎時間。プレート内のストレッサーの低濃度を使用して、酸化ストレス耐性アッセイを行う場合には、高齢化は、データの誤った解釈につながる可能性が酸化ストレス、時に動物の行動に影響を与える可能性があります。しかしながら、本明細書に記載のアッセイで、この問題は、L4 /若い成体動物を使用しているので、発生しにくいです。また、このプロトコルは、安価であり、その結果、遺伝的スクリーニングのための魅力的なこの技術をレンダリング一日で得られます。全体的に、これは、酸化ストレスに関連するヒト疾患のより良好な特徴付けに変換することができ、酸化ストレスシグナル伝達経路を理解するのに役立ちます。

比較すると、野生型C.エレガンスはflcn-1（ok975）変異動物をここでは、野生型Cの抵抗を決定するために、100 mMのPQを使用しました酸化ストレス、熱、および無酸素23に抵抗することが示されているflcn-1（ok975）動物と比較してエレガンス 。処理の4時間後、野生型の48.3パーセントはflcn-1で77.8パーセント生存率（ok975）動物と?...

Watch this Scientific Journal Video about Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates at JoVE.com

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

の酸化ストレス抵抗を測定<em&gt;線虫（Caenorhabditis elegans）</em&gt; 96ウェルのマイクロタイタープレート中

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

measuring-oxidative-stress-resistance-caenorhabditis-elegans-96-well

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Biology

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

15.3K ビュー.  McGill University. C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates. 

ビデオ: Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

計測線虫（Caenorhabditis elegans）</em固体培地上>寿命

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

生物学

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

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 ...

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 ...

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 ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

アイスキャップ：成長させる方法シロイヌナズナ</emハイスループットジェノタイピング用96ウェルプレートで>とトマト

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

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, ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

ゼブラフィッシュ胚における酸化ストレスの分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

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 ...

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 ...