Name: assessing lysosomal alkalinization in the intestine of live caenorhabditis elegans
Uploaded: 2018-04-13T14:30:00.000+00:00
Duration: 6 min 18 s
Description: Watch this Scientific Journal Video about Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans at JoVE.com

系統、自然科学工学研究会議 (レベル) に線虫の遺伝学センターとカナダ財団の資金調達の革新 (CFI) のために感謝したいと思います。博士 Exing 王 (アソシエイト ・ ディレクター、光イメージング施設と同様、博士 Lizhen 陳 (細胞工学システム、解剖学、UT 健康サン ・ アントニオ) すべての線虫の実験のための彼女の研究室設備の無制限使用を許可するために感謝したいと思います、ユタ州健康サン アントニオ) 共焦点顕微鏡による援助のため。サポートおよびビデオ撮影を容易にするために奨励を提供するため博士マイロン イグナチオに感謝も申し上げます。

1. 染色し、腸内リソソームをイメージ
<ol><li>シード線虫成長媒体 (NGM) 鋼板 OP50
	<ol>
<li>19推奨プロトコルに従って NGM 板を準備し、閉じたプレートは室温で 2 日間乾燥を許可します。</li>
		<li>滅菌ルリア スープ (LB) スープに OP50 細菌を接種する、37 ° c 36 h または外径が 0.2 〜 0.4 まで揺れインキュベーターや水のお風呂で育ちます。細菌培養を用いた OD を避ける > 1 または外径 < 0.2。</li>
		<li>一度 NGM 板が乾燥、プレートの中央に OP50 接種のドロップ (〜 30 μ L) を配置し、約 2 cm × 2 cm サイズでは、パッチにドロップを広めます。 注: 任意残り OP50 接種は 1 ヶ月最大 4 ° C で保存できます。</li>
		<li>接種に (約 10 または 15 分) が乾燥したら OP50 板を反転し、36 h の 37 ° C で版を孵化させなさい。</li>
		<li>36 時間後まで後で使用の定温器および 4 ° C でストアからプレートを削除します。 注: プレートは 4 ° C で 1 ヶ月に適してする必要があります。</li>
	</ol></li>
	<li>OP50 に cDCFDA を補う
	<ol>
<li>OP50 プレートの上に cDCFDA を補うためには、ジメチルスルホキシド (DMSO) で 10 mM cDCFDA の在庫を用意してください。光と後で使用のための-20 ° C でのストアから保護された cDCFDA のソリューションを保持します。</li>
		<li>パッチ全体が cDCFDA で均一に覆われているように、2 cm × 2 cm OP50 パッチの表面に 10 mM cDCFDA 溶液 100 μ L をそっと置きます。 注: OP50 のパッチ全体が cDCFDA で覆われていることが非常に重要です。必要な場合は、各プレートの cDCFDA の最大 150 μ L を使用します。また、cDCFDA ソリューションが OP50 パッチから流れる cDCFDA の食品に組み込まれません、染色強度縮小になりますので OP50 パッチの境界を超えて拡張していないことが不可欠です。</li>
		<li>CDCFDA ソリューションのすべての細菌のパッチに吸収されるまで、ベンチ上暗いボックスに OP50 プレートを置き (通常約 25 〜 30 分)。</li>
	</ol></li>
	<li>CDCFDA を使用してワームを染色
	<ol>
<li>CDCFDA は、OP50 パッチを浸透している完全に、一度プレートごとの以上 20 のワームを置き、14 時間の最小値のための 20 の ° C で反転板を孵化させなさい。 注: ライトへの露出を最小限に抑えるため不透明なボックスに cDCFDA プレートを配置することをお勧めします。</li>
		<li>実験の摂取量の違いを制御し、cDCFDA 信号を正規化する (推奨) 3-{2-[(1H,1'H-2,2'-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato) (共同染色フッ素) ホウ素 (たて 1 ミリメートルのソリューションの 2.5 μ L)、その蛍光は主として非変酸性 pH スペクトル acidotropic 弱いアミン染料 (試薬の一般的な名前の材料表を参照してください)。 注: これが不十分な cDCFDA 染色強度を提供し、ライソゾーム pH の誤った解釈を与えるので 14 h 未満のワームを染色しません。</li>
	</ol></li>
	<li>顕微鏡のスライドの準備
	<ol>
<li>テープに約 3 インチ ミラー (図 1) などの滑らかな平らな面に離れてラベル付けの 2 つの平行ストリップを配置します。</li>
		<li>撥水スプレーが付いているミラーの表面をコート、余分な液体をふき取る。</li>
		<li>完全に液化まで電子レンジで (蒸留 H2O に溶解) 2% の agarose を溶かし、テープの 2 つのストリップ間の agarose の 50 μ L のドロップを配置し、スライドが垂直のストリップに顕微鏡のスライドとドロップについて簡単に説明テープ。Agarose が固化した後、スライドの下の円形のパッドを形成スライドを裏返し、5 mM アジ化ナトリウム (NaN3) agarose パッド上の 10 に 15 μ L を追加します。 注: ナン3はシトクロム C 阻害剤時に低濃度で使用、可逆画像の中には移動しませんので、ワームに麻酔がかかります。</li>
		<li>CDCFDA と補われる OP50 プレートからワームを選ぶし、任意 OP50 せずきれいな NGM プレートに転送しています。ワームは、地表から削除する、OP50 のほとんどを許可する秒数移動するワームを許可し、5 mM アジ化ナトリウムを含む agarose パッドにワームを転送します。</li>
		<li>すぐにカバー スリップでアガロース パッドをカバーします。優しくこれワーム破裂で起因できるので、あまりにも多くの圧力を適用することがなくカバー スリップを配置することを確認します。 注: cDCFDA を添加した OP50 細菌蛍光顕微鏡で可視化したとき、それ故にアジ化ナトリウムを含む agarose パッドにそれらを置く前に可能な限り、ワームをきれいにすることが重要です。線虫(N2、野生型のコントロールを含む) の以上 6 系統を準備中している場合、6、ワームが agarose パッド上の乾燥しないでくださいことを確保するためのバッチのサンプルを準備することをお勧めします。いくつかの系統で、外陰部はそれに応じて計画することが重要ですので、カバー スリップからの圧力により時間の延長期間の後破裂を開始します。</li>
	</ol></li>
	<li>共焦点顕微鏡 注: いくつかの顕微鏡蛍光強度を補うため蛍光の変数のレベルが自動的に増加する組み込み機能があります。そのような顕微鏡は、サンプルの蛍光強度は増加が本質的にするので、cDCFDA で染色ワームをイメージングのためによく働かないかもしれない。<ol>
<li>励起/蛍光波長 488/530 に設定を使用して標準的な共焦点顕微鏡のイメージングを実行 cDCFDA の nm。腸内リソソームの単一の平面画像 (ない Z スタック) を取るし、強度の定量化のための焦点 (最大信号強度) は、リソソームのみを使用します。 注: おそらく染料の可用性の違いのためのすぐ後方咽頭に小腸細胞のリソソームを維持 cDCFDA 染色遺伝子型や年齢に関係なく、それゆえ注意が必要これらの細胞のリソソームをイメージングを避けるために。</li>
		<li>2 日間の古い野生型 N2 はまずワームを含むスライドをイメージします。そうしながら染色 cDCFDA が飽和状態ではないことを確保するためのレーザー出力のピンホール、絞りなど共焦点撮像パラメーターを調整します。 注: これらの設定を設定した後は変更その日の画像の全体の持続期間のため。</li>
		<li>腸 (3 に 4 枚ワーム) の単一の平面の 1024 x 1024 ピクセルの画像をキャプチャ、60 倍レンズを使用します。 注: ワームの cDCFDA の発光スペクトルは、520 nm は、報告された蛍光スペクトル20、腸蛍光 (図 3) とは異なる特定の信号読み出しを提供するとの一致で単一の顕著なピークを得られます。</li>
		<li>生共焦点画像 (.lsm ファイル) を各チャネルの .tiff ファイルとしてエクスポートします。</li>
		<li>次に、ImageJ を開き、ファイルをクリックして |開いてを定量化する画像ファイルを開きます。イメージが読み込まれると、一度 ImageJ で楕円形選択ツールを使用して、利益 (率 ROI) の領域を選択します。</li>
		<li>その後、分析をクリックして |メジャー ROI の蛍光強度を定量化します。画像 1 枚あたり 4-5 異なるフォーカスのリソソームを選択し、利益 (率 ROI) の各地域の平均の相対的な蛍光強度を計算します。</li>
		<li>各イメージのリソソーム間腸地域のバック グラウンド蛍光強度を測定します。リソゾーム蛍光強度の値からバック グラウンド蛍光強度の値を減算します。</li>
		<li>合計では、約 30 に 50 cDCFDA 強度 (6 に 10 動物) テストされる各緊張に個別正規化した値を収集します。これらの値を使用して、任意の適切な統計解析ソフトウェアを使用してボックス、ウィスカのプロットを印刷します。 注: は、蛍光強度の比較が同じ染色実験のサンプル プロセス内で関連するだけになるよう温度、培養時間、動物の全体的な健康/年齢のような要因によってかなり異なります染色できます。したがって、すべての染色実験のコントロールを処理するために重要です。統計的な差異を計算 (t-テスト)、適切な統計ソフトウェアを使用します。</li>
		<li>イメージすべては順番に世話を撮像パラメーターのいずれかを変更すること (同じ日にステンド グラス) 同じ実験から系統します。</li>
	</ol></li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomics+analysis+of+differential+protein+expression+and+oxidative+modification+of+specific+proteins+in+the+brains+of+old+mice.">Quantitative proteomics analysis of differential protein expression and oxidative modification of specific proteins in the brains of old mice.</a> Neurobiol Aging. 27 (7), 1010-1019 (2006).</li><li>Yin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+basis+of+lipofuscin,+ceroid,+and+age+pigment-like+fluorophores.">Biochemical basis of lipofuscin, ceroid, and age pigment-like fluorophores.</a> Free Radic Biol Med. 21 (6), 871-888 (1996).</li><li>Nehrke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reduction+in+intestinal+cell+pHi+due+to+loss+of+the+Caenorhabditis+elegans+Na+/H++exchanger+NHX-2+increases+life+span.">A reduction in intestinal cell pHi due to loss of the Caenorhabditis elegans Na+/H+ exchanger NHX-2 increases life span.</a> J Biol Chem. 278 (45), 44657-44666 (2003).</li><li>Chakraborty, K., Leung, K., Krishnan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+lumenal+chloride+in+the+lysosome+is+critical+for+lysosome+function.">High lumenal chloride in the lysosome is critical for lysosome function.</a> Elife. 6, (2017).</li></ol>

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高齢化に貢献するさまざまな細胞および分子イベント生活史特性と遺伝的要因によって影響を受けます。私たちの最近の研究22では、生殖周期がライソゾーム pH の動態の調節を介して相馬のフィットネスの制御において重要な役割を果たしていることを示唆しています。V ATPase の転写は、順番により、酸性リソソームのアップレギュレーションによって動物を積極的に再現?...

タンパク質凝集体の外観広く認められる真核細胞1,2,3と細胞老化4原則ドライバーの中に思想が形成の高齢化の特徴,5,6,7. 年齢を細胞、タンパク質の異化が障害、タンパク質凝集の増加につながる成長証拠があります。老化細胞における蛋白質分解の破綻には、プロテアソームを介したタンパク質分解9と同様、オートファジー8の障害が含まれます。最後に、古い細胞の増加は不可逆的なタンパク質の酸化タンパク質異化10をさらに損なうこと。
Autophagy は当初一括劣化損傷を受けたタンパク質の非選択性プロセスであると考えられたが、最近の研究は、オートファジーはタンパク質凝集体とは細胞小器官の機能不全の非常に選択が示されています。他の蛋白質のクリアランス機構11による特性劣化を受けやすい。オートファジーのプロセス中に破損したと集計されたタンパク質は、オートファゴソームと呼ばれる二重膜の小胞に隔離します。このオートファゴソームは、オートファゴソーム貨物12の劣化につながる、リソソームと呼ばれる酸性の細胞内小器官と融合します。リソソーム オートファジー経路のエンドポイントを表し、細胞膜修復、転写調節、栄養リモートセンシングなど別の細胞プロセスに参加(文献 13 見直し) のホメオスタシスで彼らの中心的な役割を強調します。いくつかの研究は、ライソゾーム機能、年齢依存的な低下と様々 な神経変性疾患13間の関連を示しています。一貫して、古い細胞のライソゾームの機能を復元すると、高齢化に関連した表現14,15の発症を遅らせることができます。Intralumen 環境の組成の研究では、古い細胞のライソゾームの機能の崩壊はリソソームタンパク16の生産の減少のためではないことを示唆しています。また、intralysosomal 酸味、その酵素活性の重要な要件の損失可能性がありますリソソームを介したタンパク質分解17でドロップの根底にあることが提案されている.この仮説を検証することができる、試薬およびレプリケート可能で一貫性のある方法で生きた細胞のライソゾーム pH のダイナミックな変化をプローブするためのプロトコルの開発が不可欠です。
線虫 c. エレガンスの腸みみずの主要な代謝組織であり、全身の恒常性と寿命の重要な調節因子。変化を評価するアッセイを開発したどのようにリソソームを介したタンパク質分解を決定するワームの腸内リソソーム内腔の酸性度で高齢化に貢献します。Hasn't されてまだ、体内ライソゾーム pH18.の小さい増加を検出する成功したプロトコルを確立する努力が pH に敏感な fluorophores が付いては、腸内のリソソームをマークする線虫で従来使用されてきた、ここでは、OP50 食品に pH に依存した蛍光体 (cDCFDA) が組み込まれています簡単で便利な餌プロトコルを使用してc. の elegansの腸の細胞のライソゾーム酸性度の損失を検出する使用できるプロトコルを提供します。

<table><tbody><tr><td>OP50 (&lt;em&gt;E. coli&lt;/em&gt;)</td><td>Caenorhabditis Genetics Center</td><td></td><td>Order online at https://cgc.umn.edu/strain/OP50</td></tr><tr><td>5(6)-carboxy-2&amp;rsquo;,7&amp;rsquo;-dichlorofluorescein diacetate&amp;nbsp;</td><td>ThermoFisher</td><td>C369</td><td>Commonly known as cDCFDA</td></tr><tr><td>9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#039;H-2,2&#039;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron</td><td>ThermoFisher</td><td>L7528</td><td>Commonly known as Lysotracker Red</td></tr><tr><td>Confocal microscope (e.g. Zeiss LSM 510)</td><td></td><td></td><td></td></tr><tr><td>ImageJ</td><td></td><td></td><td>Download for free from https://imagej.nih.gov/ij/download.html</td></tr><tr><td>LB Broth powder</td><td>ThermoFisher</td><td>22700041</td><td></td></tr><tr><td>Bacto Agar</td><td>Sigma</td><td>A5306-1KG</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>Bacto Peptone</td><td>Fisher Scientific</td><td>S71604</td><td></td></tr><tr><td>Cholesterol powder</td><td>Sigma</td><td>C3045&amp;nbsp;</td><td></td></tr><tr><td>CaCl2</td><td>Sigma</td><td>449709</td><td></td></tr><tr><td>MgSO4</td><td>Sigma</td><td>M7506</td><td></td></tr><tr><td>K3PO4</td><td>Sigma</td><td>P5629</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma</td><td>S2002</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td></td></tr><tr><td>Microscope Slides</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Cover Slips</td><td>ThermoFisher</td><td>3406</td><td></td></tr><tr><td>Agarose</td><td>Sigma</td><td>A6013</td><td></td></tr><tr><td>Incubator</td><td></td><td></td><td></td></tr><tr><td>Mirror or other smooth flat surface</td><td></td><td></td><td></td></tr></tbody></table>

assessing lysosomal alkalinization in the intestine of live caenorhabditis elegans

線虫線虫(C. elegans) は、長寿と発達経路の研究に広く使用されているモデル システムです。このような研究は動物、転送し逆遺伝学的アッセイ、生成の蛍光に分類された蛋白質の相対的な容易さと初期に microinjected することができますいずれか蛍光染料の使用能力の透明物によって促進されます。胚、または細胞細胞器官 (例えば9-ジエチルアミノヒドロキシベンゾイル-5 H-ベンゾ (a) 誘導 5-1 と (3-{2-[(1H,1'H-2,2'-bipyrrol-5-yl-kappaN(1)) methylidene]-2 H-pyrrol-5-イル-活版} - N は - のラベルにその食品(大腸菌 OP50)に組み込まれました。[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron)。今回腸内リソソームを汚れ蛍光 pH 感受性色素の使用ライブ ワームのライソゾーム酸性度の動的な生理学的な変更のビジュアルの読み出しを提供します。このプロトコルはリソソームの pH を測定しないが、むしろライソゾーム酸性度の生理学的な関連語句を評価する信頼性の高い方法を確立することを目的と。cDCFDA は細胞内のエステラーゼによって加水分解時に蛍光蛍光 5-(and-6)-carboxy-2',7'-dichlorofluorescein (cDCF) に変換はセル側の透過物化合物です。リソソーム内のプロトン化それが蓄積する場所、これらのオルガネラの cDCF をトラップします。4.8 その低い pKa、ためこの染料は、酵母の pH センサーとして使用されています。ここで腸内リソソーム内の酸性度を評価するために栄養補助食品として cDCFDA の使用を説明線虫。この技術は生きている動物,に取って代わるリソソームの発見が可能、老化, オートファジー, およびリソソーム器官に関する研究を含む実験的アプリケーションの広い範囲を持っています。

cDCFDA は pH 依存的にリソソームを汚れ、その低い pKa およびリソソームに準備ができて吸収理想的な pH センサー21になります。cDCFDA 染色、ライソゾーム pH (pH が下がると強度の増加が染色されているすなわち)18,22に反比例します。cDCFDA 信号が v-atp アーゼ プロトン インポート22 に必要な...

Watch this Scientific Journal Video about Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans at JoVE.com

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

PH に敏感な重要な染料 6 - カルボキシ - を使用して 2', 7'-ジクロルフルオレッセン アセテート (cDCFDA) のc. の elegansの腸のライソゾーム酸性度の損失をプローブするステップ バイ ステップ ガイド

腸の生きている線虫のライソゾーム アルカリ化を評価します。

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

assessing-lysosomal-alkalinization-intestine-live-caenorhabditis

Research

JoVE Journal

Developmental Biology

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

5.9K ビュー.  University of Saskatchewan. PH に敏感な重要な染料 6 - カルボキシ - を使用して 2', 7'-ジクロルフルオレッセン アセテート (cDCFDA) のc. の elegansの腸のライソゾーム酸性度の損失をプローブするステップ バイ ステップ ガイド

ビデオ: Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

小規模な比色試金の細胞内乳酸と線虫の線虫のピルビン酸

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

生化学

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

細胞間腔の形態形成と単一細胞レベルでのIn Vivo偏光膜生合成のためのモデルとして線虫腸: 抗体の汚損、RNAi 損失の機能解析、イメージングによるラベリング

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

発生生物学

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

内脂肪量の生化学的および高スループット微視的評価<em&gt;線虫（Caenorhabditis elegans）</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

生物学

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 ...

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.

ケネラブディティス・エレガンスの腸透過性に及ぼす細菌および化学物質の影響の測定

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a ...

医学

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

インビボカエノハブディティス・エレガンスニューロンにおける微小管動態と向きの評価

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...

神経科学

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

筋肉の細胞内コンパートメントを評価するための方法<em&gt; C。エレガンス</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

脂質豊かさとナイルの赤とオイル赤 O 染色により線虫の脂質分布評価の定量化

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

刺激されたラマン散乱顕微鏡を用いた カエノラム型エレガンス における脂質蓄積ダイナミクスの無標識イメージング

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

<meta charset="utf8"></head><body>線虫Caenorhabditis elegansの代謝と老化パターンの評価

Using Low-Cost Dyes to Visualize Glycogen Accumulation and Gut Integrity in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) is a transparent, non-parasitic nematode with a simple biology, which makes it a great tool for biological sciences teaching through the staining of the cells or their molecular content. Lugol dye (iodine-potassium iodide solution) has been widely used in biochemistry to stain glycogen stores. In this context, it is possible to observe differences between fed and starved animals, besides the effects of different conditions, such as different diets and oxygen levels. Erioglaucine is a blue dye that indicates the loss of the intestinal barrier. When the intestinal barrier is intact, the blue dye stains inside the lumen; however, when this integrity is disrupted, the dye leaks into the body cavity. Using a stereomicroscope or a microscope, teachers can demonstrate physiological and biochemical alterations, or they can instigate students to ask a scientific question and hypothesize and test their hypothesis using these assays. The present protocol describes two staining techniques in C. elegans that can be easily carried out by students.

<meta charset="utf8"></head><body>著者スポットライト:低コストでインパクトのあるアッセイを使用した C.エレガンスの 代謝および老化プロセスの探索

Teaching biological sciences can be made more stimulating for students through the use of experimentation. This manuscript presents two different yet complementary protocols that can be utilized in the classroom to encourage students to formulate and test hypotheses related to high-calorie diets, starvation, and aging.

低コストの染料を使用して、線虫のグリコーゲン蓄積と腸の完全性を視覚化する

Caenorhabditis elegans (C. elegans) is a transparent, non-parasitic nematode with a simple biology, which makes it a great tool for biological sciences ...

腸の生きている線虫のライソゾームアルカリ化を評価します。

要約

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この動画の章

小規模な比色試金の細胞内乳酸と線虫の線虫のピルビン酸

細胞間腔の形態形成と単一細胞レベルでのIn Vivo偏光膜生合成のためのモデルとして線虫腸: 抗体の汚損、RNAi 損失の機能解析、イメージングによるラベリング

内脂肪量の生化学的および高スループット微視的評価

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

ケネラブディティス・エレガンスの腸透過性に及ぼす細菌および化学物質の影響の測定

インビボカエノハブディティス・エレガンスニューロンにおける微小管動態と向きの評価

筋肉の細胞内コンパートメントを評価するための方法

脂質豊かさとナイルの赤とオイル赤 O 染色により線虫の脂質分布評価の定量化

刺激されたラマン散乱顕微鏡を用いたカエノラム型エレガンスにおける脂質蓄積ダイナミクスの無標識イメージング

低コストの染料を使用して、線虫のグリコーゲン蓄積と腸の完全性を視覚化する

腸の生きている線虫のライソゾーム アルカリ化を評価します。

要約

さらに動画を探す

この動画の章

小規模な比色試金の細胞内乳酸と線虫の線虫のピルビン酸

細胞間腔の形態形成と単一細胞レベルでのIn Vivo偏光膜生合成のためのモデルとして線虫腸: 抗体の汚損、RNAi 損失の機能解析、イメージングによるラベリング

内脂肪量の生化学的および高スループット微視的評価

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

ケネラブディティス・エレガンスの腸透過性に及ぼす細菌および化学物質の影響の測定

インビボカエノハブディティス・エレガンスニューロンにおける微小管動態と向きの評価

筋肉の細胞内コンパートメントを評価するための方法

脂質豊かさとナイルの赤とオイル赤 O 染色により線虫の脂質分布評価の定量化

刺激されたラマン散乱顕微鏡を用いた カエノラム型エレガンス における脂質蓄積ダイナミクスの無標識イメージング

低コストの染料を使用して、線虫のグリコーゲン蓄積と腸の完全性を視覚化する

腸の生きている線虫のライソゾームアルカリ化を評価します。

刺激されたラマン散乱顕微鏡を用いたカエノラム型エレガンスにおける脂質蓄積ダイナミクスの無標識イメージング