作者はこのメソッドの最初の基礎のためにジョンウルフォードに感謝、TGKはGM77073によってサポートされているNIH GM057483とAI076245とPRCでサポートされています。

1。 7から47パーセントの蔗糖勾配の準備<ol><li>勾配が連続になることを可能にするために使用する前に一日蔗糖勾配を準備します。 7％滅菌作成し、4で50mmのNH 4 Clを含む47％ショ糖溶液、50mMトリス-酢酸のpHは7.0と12mmのMgCl 2と店舗℃で1。 </li><li> 6勾配は、以下のショ糖濃度の各48 mLを作るために7％、47％ショ糖ストック溶液を混合するため。 1mMの最終濃度がそれぞれのショ糖溶液にDTTを（1M株式）を追加します。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 最終濃度 </td><td> 7％のスクロース </td><td> 47％ショ糖 </td></tr><tr><td> 7パーセント</td><td> 48 mLの</td><td> 0 </td></tr><tr><td> 17パーセント</td><td> 36 mLの</td><td> 12mlの</td></tr><tr><td> 27パーセント</td><td> 24 mlの</td><td> 24 mlの</td></tr><tr><td> 37パーセント</td><td> 12mlの</td><td> 36 mLの</td></tr><tr><td> 47パーセント</td><td> 0 </td><td> 48 mLの</td></tr></tbody></table></li><li>勾配を注ぐためには、確保してパラフィルムで封で20 mLシリンジにパスツールピペットを添付したり、長い針を使用してください。 1 × 3.5&quot;ポリアロマー超遠心チューブの底に7％のショ糖の7 mLのを追加します。次は、チューブの底近くにパスツールピペットの先端を配置しても高速な0.3よりをピペッティングしないで7％のソリューションの下に17％ショ糖溶液の7 mLを加えml /秒。7 mLの27％の各々と繰り返し、37％および47％ショ糖溶液。あなたはレイヤーの間に明確な線を守るべき、最低限の混合はボトル、ローターと一緒に℃で一晩4℃。ストアの勾配が発生したことを示すとサンプルを収集するために使用されるチューブ。 </li></ol> 2。酵母エキスの調製<ol><li> 0.8から1.0のOD 600に酵母培養液125 mLを成長する。 100μg/ mlの終濃度に培養液にシクロヘキシミドを追加し、30で振とう℃で15分間続ける。 </li><li>一方、80μg/ mLのシクロヘキシミド、200μg/ mLのヘパリン、0.2％DEPC、および10mMのトリス-塩酸pH7.5、0.1MのNaCl、30mMのMgCl 2を含む溶解緩衝液を準備。この時点から氷の上にすべてをしてください。 </li><li> 250mLの遠心ボトルに文化を注ぎ、氷を詰めます。 4℃で5分間8000 × gで（SLA - 1500ローターで7250 rpm）で遠心する氷冷溶解緩衝液と15 mLポリプロピレンチューブに転送10mLで一回ペレットを洗浄します。 4℃で3分間℃で5900 × gで（SLA - 600TCローターで〜6,000 rpm）で遠心する、溶解バッファー0.5 mlにペレットを1.5mlチューブへの転送やチルド、焼きたての酸洗浄ガラスビーズ0.5 mLを加える。渦four 30秒間隔のための細胞、各インターバルの間に30秒間氷上でチューブを冷却する。 1.5 mlチューブに、溶解緩衝液の別の0.5 mLを追加します。で10分間4℃で遠心の最高速度（エッペンドルフ5417Rの遠心で14,000 rpm）で遠心する新しい1.5 mlのマイクロ遠心チューブに上清を移す。 OD 260 / OD 280の比率を決定するために別のチューブに10μlを削除する（下のステップ3.1を参照）。 -80℃ストアエキス℃の</li></ol> 3。哺乳動物細胞からの抽出物の調製<ol><li>接着細胞の場合は、〜70％コンフルエントに100 mmディッシュで育つ。あなたは11 mlの勾配ごとに100 mmディッシュ（典型的な収率は〜20 OD 260単位です）が必要となります。大きくしたり小さく勾配の大きさのために直線的に量を拡大縮小します。非接着細胞の場合、約1 × 10 7細胞を11 mlの勾配ごとに必要とされる。 </li><li>溶解の前に、100μg/ mLとするシクロヘキシミドを加える。 37℃で15分間インキュベートする。氷にプレートを移し、氷冷PBSで細胞を2回すすいでください。将来のすべてのステップは0〜4℃で実施されなければならない（MUST） </li><li>吸引によるPBSの痕跡をすべて削除する（プレートは氷の傾斜ベッドの上で排出させる）と1 mlの溶解バッファーを追加し、あらかじめ冷却した1.5​​mlチューブにスクレープして転送。溶解バッファーの成分は、細胞の種類や成長条件を最適化することが必要になります。良い出発バッファーは20mMのHEPES - KOH、pH7.4の15 mMのMgCl 2、200mMのKClを、1％トリトンX - 100（V / V）、100μg/ mlのシクロヘキシミド、2mMのDTT、および1 mg /になりますmLのヘパリン。主要な変数は、細胞骨格や膜結合ポリソームの抽出を向上させるためにマグネシウムと塩化カリウムの濃度だけでなく、非イオン性界面活性剤の包含または除外です。二回27ゲージの注射針を通過したり、タイプB乳棒を使用してダウンスの均質化の5-10ストロークによって細胞を溶解する。注射器やホモジナイザーは使用前にあらかじめ冷却してください。 </li><li>冷蔵微量遠心機で5分間、14,000 xgでスピン。後で使用するために液体窒素でのショ糖密度勾配（哺乳動物細胞のためにこれをトリトンX - 100およびヘパリンが、それ以外は1.2と1.3で説明されているように欠けている溶解バッファーで構成されている）やフラッシュ凍結に新鮮なライセートを転送する。 </li></ol&gt; 4。勾配の遠心分離<ol><li>氷の上にポリリボソームライセートを解凍。水1mLに予約されて10μLのサンプルを追加し、分光光度計でOD 260 / OD 280の比率を読み取ることによって、溶解物中の核酸の濃度を決定する。説明されているように成長酵母培養液からの典型的な収率は50 OD単位です。 </li><li>勾配の上に層ライセートの25 OD 260ユニットを静かに、勾配の表面近傍の管の壁に置かれたチップを用いてピペットを使用する。用溶解液の35 mLの勾配を500μlは一般的にロードされます。溶解バッファーは、すべての勾配が等しいボリュームがロードされるように使うことができます。材料の下の量は、より良い分解能が得られます。鉗子を使用して、慎重にスイングロータのバケットに勾配を下げる。 </li><li> 4℃で4時間、100,000 × gで（Surespin 630ローターで23000 rpm）で勾配を遠心℃にこのプロトコルは、より小さい容積の勾配に適合させることができる。 </li></ol> 5。データ及び分画のコレクション<ol><li>前の4時間のスピンの完了まで約30分には、モデル184チューブのピアサーに針を取り付けます。オンラインイスコモデルUA - 5の吸光度/蛍光モニターにペンを取り付けます。前の分析試料に到達するために安定したベースラインを可能にするために吸光度をモニターの電源を入れます。ポリソームプロファイルの電子買収を簡単にチャートレコーダにDI - 148Uのデータ収集ユニット（DATAQインスツルメンツ）を取り付けることにより得ることができます。プロファイルは、リアルタイムで収集し、付属WinDaqのソフトウェアを使用して、後で注釈のために保存することができます。 </li><li>フロリナート逆流、迅速な設定を使用しての50mLのシリンジポンプを埋める。現在に気泡がないことを確認してください。注射器からチューブのピアサーにホースを接続し、簡単に、気泡のホースをクリアするために順方向に3mL /分でフロリナートの流れをオンにします。 </li><li>サンプルを収集するフローセルの最後にホースの下に分画を集めるがオフ実行する場合はチューブの廃棄物のビーカーまたはシリーズを置きます。 </li><li>慎重に遠心ローターからショ糖勾配を含むポリアロマー遠心分離管を削除し、（不穏な勾配を避けるために、空のチューブと氷の井戸を事前に形成する）氷の上に置きます。 </li><li>細胞抽出物を分析するために、管の穿孔に勾配管を配置。コンセントにチューブの上部を接続し、固定します。針がチューブの底を貫通するように遠心分離管に底のステージを上げる。針は、遠心分離管の底部から突出されると、6mL/minで順方向にフロリナートの流れを開始する。 </li><li>フロリナートはチューブに流入開始すると、オンラインの吸光度をモニター上の針の動きを監視する。針が動き始めると、60の速度設定にチャートの動きをオンにします。モニターは、40Sリボソームサブユニットの検出およびポリリボソーム複合体まで続くが続くフリー材料で始まるOD 254を記録します。あなたがRNAやタンパク質の解析のための分画を収集している場合、通常0.2分の画分を（1mL）を取られる。 RNA解析のために、画分を抽出する好ましい方法に応じて、トリゾール（Invitrogen社製）、フェノールまたはSDS / Proteinase Kを含むチューブに直接収集する必要があります。 </li><li>ポリリボソームプロファイルの末尾に達したときに、遠心分離管にフロリナートの流れをオフにして、吸光度/蛍光モニターのチャートの動きを止める。 </li><li>注射器にチューブからショ糖を描画しないように確実に速い速度で逆方向の流れを開始することにより、シリンジへの遠心管からフロリナートを引っ込めます。チューブのピアサーから緩めて遠心管には、針を下げ、そしてチューブを取り外します。 </li><li>繰り返して、各スクロース勾配5.8を介して5.5を繰り返します。 </li><li>すべての蔗糖勾配が分析されているときは、注射器に戻ってホースからフロリナートを引っ込める。上部にある廃棄物のホースを含む分留塔の各部分を（管の穿孔とフローセル）を外し、水でよくすすいでください。 </li></ol> 6。代表的な結果<img src="/files/ftp_upload/1948/1948fig1.jpg" alt="図1" /> 図1。リボソーム抽出の代表的なトレースがシクロヘキシミドの存在下で酵母株BY4741（ マットHIS3Δ1 LEU2Δ0 met15Δ0 URA3Δ0）から調製した。リボソームエキスは7 47％のショ糖勾配を利用して分画し、UV検出器に接続されたポンプのシリンジ装置を用いて分析した。 polyribosomesと80S、60Sと40Sリボソームを含むピークが示されている。

<ol>
	<li>Baim, S. B., Pietras, D. F., Eustice, D. C., Sherman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+allowing+an+mRNA+secondary+structure+diminishes+translation+of+Saccharomyces+cerevisiae+iso-1-cytochrome+C.">A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome C.</a> Mol. Cell. Biol. 5, 1839-1846 (1985).</li><li>Ripmaster, T. L., Vaughn, G. P., Woolford, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DRS1+to+DRS7,+novel+genes+required+for+ribosome+assembly+and+function+in+Saccharomyces+cerevisiae.">DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.</a> Mol. Cell. Biol. 13, 7901-7912 (1993).</li><li>Anand, M., Chakraburtty, K., Marton, M. J., Hinnebusch, A. G., Kinzy, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interactions+between+yeast+translation+eukaryotic+elongation+factor+(eEF)+1A+and+eEF3.">Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3.</a> J Biol Chem. 278, 6985-6991 (2003).</li><li>Serikawa, K. 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+transcriptome+and+its+translation+during+recovery+from+cell+cycle+arrest+in+Saccharomyces+cerevisiae.">The transcriptome and its translation during recovery from cell cycle arrest in Saccharomyces cerevisiae.</a> Mol Cell Proteomics. 2, 191-204 (2003).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324, 218-223 (2009).</li><li>Valasek, L., Szamecz, B., Hinnebusch, A. G., Nielsen, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+stabilization+of+preinitiation+complexes+by+formaldehyde+cross-linking.">In vivo stabilization of preinitiation complexes by formaldehyde cross-linking.</a> Methods Enzymol. 429, 163-183 (2007).</li><li>Fletcher, J. E., Copeland, P. R., Driscoll, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+distribution+of+phospholipid+hydroperoxide+glutathione+peroxidase+mRNA:+evidence+for+a+block+in+elongation+at+the+UGA/selenocysteine+codon.">Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon.</a> Rna. 6, 1573-1584 (2000).</li><li>Ruan, H. J., Brown, C. Y., Morris, D. R., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of mRNA formation and function. , 305-321 (1997).</li><li>Martin, G. W., Berry, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selenocysteine+codons+decrease+polysome+association+on+endogenous+selenoprotein+mRNAs.">Selenocysteine codons decrease polysome association on endogenous selenoprotein mRNAs.</a> Genes Cells. 6, 121-129 (2001).</li><li>Lee, Y. Y., Cevallos, R. C., Jan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+upstream+open+reading+frame+regulates+translation+of+GADD34+during+cellular+stresses+that+induce+eIF2alpha+phosphorylation.">An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation.</a> J Biol Chem. 284, 6661-6673 (2009).</li></ol>

利害の衝突は宣言されません。

ポリリボソームプロファイルから得られる情報は、セルの並進状態に貴重な洞察を提供することができます。さらに、リボソームサブユニット自体のアセンブリの状態は、2を決定することができます。例えば、プロフィール上で右にわずかに肩を持つhalfmersまたは80Sと大きなピークの存在は、結合した40Sが待っている60Sが参加するサブユニットがサブユニットを示しています。伸びのいずれかを追加シクロヘキシミドまたは他のインヒビターの不在下で実験を実行すると、伸びが3に変更されているかどうかを示す流出の速度、分析することができます。分数自体は分数の北部やウェスタンブロッティングによってリボソームサブポピュレーションを持つ特定のmRNAまたはタンパク質の協会のその後の測定用試薬の豊富な情報源です。アクティブリボソームに関連付けられているmRNAの合計プールが関連付けられているマイクロアレイ4または深いシーケンシング解析5を介して決定することができます。蛋白質の関連付けも試薬6を架橋の適当な添加によって安定化させることができる。 哺乳動物細胞からポリリボソーム解析にも安定したポリソームを得るために必要な条件を確立することで見かけの難しさの点で異なるプロトコルとして言及する価値がある。文献に報告されている緩衝液系は、7-10広く可変であるため、溶解バッファーの細胞型に特異的な最適化のための要件があるかもしれません、または、バッファシステムは、作業への高い関心と同じくらい重要ではないことも同様に妥当である新鮮なライセートは、低温で保管。我々の知る限りでは哺乳類のポリソーム形成に影響を与えるパラメータの体系的な評価が報告されていない。

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>polyallomer ultracentrifuge tube</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>326823</td>
<td>1 x 3.5 in. (25x89mm)</td>
</tr>
<tr>
<td>Fluorinert</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F9755</td>
<td>&nbsp;</td>
<tr>
<td>Cycloheximde</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C-7698</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3393</td>
<td>&nbsp;</td>
<tr>
<td>BY4741</td>
<td>&nbsp;</td>
<td>Open Biosystems</td>
<td>YSC1048</td>
<td>Other strains work equally well</td>
</tr>		</tbody>
		</table>
		</div>

eukaryotic polyribosome profile analysis

タンパク質合成は、様々なレベルで制御されている複雑な細胞プロセスです。例えば、グローバルな翻訳は、アミノ酸飢餓や成長因子の撤退などの細胞ストレスの種々の方法によって開始フェーズまたは伸長段階で抑制することができる。また、個々のmRNAの翻訳は、mRNA局在や同種のマイクロRNAの存在によって調整することができる。タンパク質合成の研究は、頻繁に翻訳調節やタンパク質合成に欠陥のメカニズムに光を当てるためにポリリボソーム解析を利用しています。このアッセイでは、mRNA /リボソーム複合体は、真核細胞から単離されている。ショ糖密度勾配は、単一のリボソームまたは一染色体に結合したmRNAからpolyribosomesと呼ばれる複数のリボソームに結合したmRNAを分離します。勾配の分画は、異なるリボソーム集団とそれに関連するmRNAまたはタンパク質の分離と定量化が可能になります。定義された条件の下でmonosomesへpolyribosomesの比率の違いは、翻訳開始または伸長/終了のどちらかに欠陥の可能性を示唆しています。ポリリボソーム画分に存在するmRNAの検討では、個々のmRNAのコホートは、実​​験条件に変更が翻訳されているかどうかを明らかにすることができます。さらに、リボソームアセンブリも勾配によって分離され、大小のリボソームサブユニットのピークの分析によってモニターすることができる。このビデオでは、我々は、酵母細胞、ショ糖勾配による抽出物の分離や結果の解釈から得られた粗リボソーム抽出物の調製方法を提示する。この手順では、哺乳動物細胞に容易に適応可能である。

Watch this Scientific Journal Video about Eukaryotic Polyribosome Profile Analysis at JoVE.com

Eukaryotic Polyribosome Profile Analysis

この記事では、真核細胞からリボソームを翻訳するの抽出のためのプロトコルについて説明します。解凍したら、リボソームが異なるリボソーム集団を分析できるようにショ糖密度勾配分画によってmonosomesとpolyribosomesに分かれています。このように、このメソッドは、翻訳の調節を調べるためのゴールドスタンダードです。

真核生物のポリリボソームプロファイル解析

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

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JoVE Journal

Biology

52.5K ビュー.  University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School. この記事では、真核細胞からリボソームを翻訳するの抽出のためのプロトコルについて説明します。解凍したら、リボソームが異なるリボソーム集団を分析できるようにショ糖密度勾配分画によってmonosomesとpolyribosomesに分かれています。このように、このメソッドは、翻訳の調節を調べるためのゴールドスタンダードです。

ビデオ: Eukaryotic Polyribosome Profile Analysis

Immunoblot Analysis

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

イムノブロット解析

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the ...

生物学

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

神経毒性の高いコンテンツ分析のための神経とアストロサイト共培養アッセイ

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

C2C12筋芽細胞を用いた生体エネルギープロファイル実験

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.

マイクロインジェクション分子およびそれらの真核生物の細胞内ターゲットを視覚化するツールに相関光学および電子顕微鏡（クレム）


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter 1. These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms 2.
Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling 3 and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms 4, 2. Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web 5. Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism 6, 7. Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited 8, 4, 9, 10, 5. A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle.
We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity 4, 11, as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms.
RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs 12. In this study, we applied a radioisotope assay modified for filtered samples 13 to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.

Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.

化学的に成層南極湖と炭素固定ポテンシャルの評価から微生物真核生物濃縮文化の確立

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

ゼブラフィッシュ胚の代謝プロファイル解析

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

における代謝プロファイルのリアルタイム分析<em&gt;エクスビボ</em&gt;マウス腸陰窩オルガノイド培養

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

真核細胞から体外の環状DNAのゲノムワイドな精製

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

水生動物使用グループ固有の rDNA プライマーの遺伝的腸内容分析の研究プロトコル

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

原核生物と真核生物の化学形態分析走査電子顕微鏡 (SEM) での乾燥の準備

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...