この作品は、NIHの国立心臓、肺、血液研究所の助成金RO1 HL81248によって資金を供給された。

1。ウシ大動脈内皮細胞（BAEC）の培養<ol><li>適切な無菌テクニックが続いていた。 </li><li> 37抗生物質なしで水浴、暖かい培地℃で</li></ol> 注：培地は4.5 g / LのD-グルコースとフェノールフリーダルベッコ改変イーグル培地（DMEM）で構成されており、10％ウシ胎児血清（FBS）および2.5を添加した4 mM L-グルタミン、1％非必須アミノ酸、 mg / Lの内皮細胞増殖因子。 <ol start="3"><li>インキュベーターからT75フラスコを含むセルを削除し、フードの内側に配置する前に、70％エタノールでフラスコの表面を清掃してください。 </li><li>アスピレーターを使用して、古い培地を除去し、ダルベッコのリン酸緩衝生理食塩水（DPBS）の5 mlで2回洗浄する。 </li><li>トリプシンの2ミリリットルを追加し、定期的に顕微鏡下で検査しながら切断する細胞4-5分間待ちます。 </li><li>培地3mlを追加して、繰り返して、別のtにピペットを用いて混在させる彼は細胞とも懸濁液を作成します。 </li><li> 5分間121グラムで15 mlのチューブと遠心分離機にトリプシンで培地5mlを転送します。 </li><li>アスピレーターを用いて上清を取り除きます。 DPBS 5mlを加え、よく混ぜます。 5分間121グラムで遠心します。 </li><li>上清を吸引除去し、培地6mlを加えることによって細胞ペレットを再懸濁する。 </li><li> 6ウェルプレートに、ウェルを培地1mlを追加するたびに細胞懸濁液1 mlを加える。ピペットを用いて懸濁液を混ぜる。 </li><li>ラベルプレートと37℃、5％CO 2を一晩インキュベートする。 </li></ol> 2A。 BAEC細胞とNOの検出<ol><li>適切な無菌テクニックが続いていた。 </li><li>インキュベーターから6ウェルプレートを取り外し、最初のウェルから培地を吸引除去する。 1 mlのDPBSで細胞を2回洗浄します。 </li><li> 1.9 mMの硫酸鉄（II）七水和物（のFeSO 4·7H 2 O、で0.8 mgを溶解することによって新たに準備する210μlを加え1 mlのCaCl 2とMgCl 2を持つDPBS）とアンモニウム210μlの午前1時07分の比を用いてN-メチル-D-グルカミンジチオカルバメート（MGD、CaCl 2とMgCl 2を 500μlのDPBSで2.7 mgを溶解することにより、新たに調製） （注：過剰MGDが使用されているこの比率は、従来のFe 3の収率は+-MGDを過剰MGDの存在下で最大化されているという事実に起因+ MGD複雑なのFe 2準備のために採用されているのに加え。 NO-のFe 3 +が -MGD形成されたので、+-MGDは必要ありませんFe 2 +を安定させるために溶液中でアスコルビン酸は内因性アスコルビン酸、ヒドロキノン、または最大の変換効率を持つシステイン+ MGD EPR検出可能なNO-Feの2に削減され99.9％...のFe 2低スピン反磁性の状態が+ NO）低温装置を必要とせずにフラットセルまたはキャピラリーチューブを用いた 15 の検出することができます 。 </li><li>よくスワール得られた懸濁液を、4.6μを追加するカルシウムイオノフォアのL（CAI）（1ミリリットルのDMSOに1mgを溶解することにより1.9 mMのストック溶液から調製）。 </li><li>スワール再びソリューションと37℃36分のためにCをさらにNO-Feの3の削減を可能にするためには、EPR検出可能なNO-Fe 2 +の -MGDに+ MGD。 </li><li>エッペンドルフチュー​​ブとEPRフラットセル（または50μlのキャピラリーチューブへの）への転送の上清（425μl）を収集します。 </li><li> EPR取得パラメータは以下のとおりです。マイクロ波周波数：9.8 GHzの、中央のフィールド：3427 G;変調振幅：6.0 G、掃引幅：100 G;レシーバゲイン：1×10 5、マイクロ波電力：10 mWで、スキャンの総数：121;掃引時間：10秒、および時定数：20ミリ秒（注：パラメータはひとつの楽器と実験条件ごとに異なりますので、したがって、唯一のセンターフィールド周波数と変調振幅が考慮すべき最も重要なパラメータである。） </li><li>室温でのスペクトルを記録し、2次元スペクトルは、INTEGたブルカーWinEPRデータ処理ソフトウェアやその他のデータ処理ソフトウェアを用いて補正し、バックグラウンドノイズやベースラインを減らすために評価した。付加体形成の定量化については、濃度対信号強度（又は面積）の標準的なプロットは、NOドナーSNAPを使用しない構築することができます。 </li></ol> 2B。 eNOSのアンカップリング実験<ol><li>インキュベーターから6ウェルプレートを取り外し、第2ウェルから培地を吸引し、1 mlのDPBSで2回洗浄する。 </li><li>ペルオキシナイトライトのドナー、5 -アミノ-3 - （4 -モルホリニル）-1,2,3 - oxadiazolium塩化物（SIN-1）はeNOSの16を切り離すために使用された。 0.5mMのSIN-1（M R 206.6たてないのCa / Mgのイオンを含むPBS 500μlの1 mgのSIN-1を溶解して調製10mMストック溶液からg /モル）、希に2ミリリットルのDPBSの100μlを加えおよび10％FBS。 </li><li> 37℃で2時間インキュベート℃、5％CO 2。 </li><li>インキュベーターからプレートを削除し、DPBSで2回洗浄する。 </li><li> 210μlを加え2.8mmでのFeSO 4·7H 2 O、19.6 mMの210μlを新たに上記の手順に従って調製MGD。 </li><li>渦解と1.9 mMのCAIの4.6μlを追加します。 </li><li>スワール再びソリューションと36分間37℃でインキュベートする。 </li><li>エッペンドルフチュー​​ブとEPRフラットセルへの転送の上清（425μl）を収集します。 </li><li> EPR取得パラメータは以下のとおりです。マイクロ波周波数：9.8 GHzの、中央のフィールド：3427 G;変調振幅：6 G;掃引幅：100 G;レシーバゲイン：1×10 5、マイクロ波電力：10 mWで、スキャンの総数：121;掃引時間：10秒、および一定時間：20ミリ秒。 </li><li>スペクトルを記録し、2次元スペクトルは、前述したように補正し、バックグラウンドノイズやベースラインを減らすために統合されました。 </li></ol> 3。 O 2の検出• -多形核好中球からの（多形核白血球） <ol><li>以前に17に記載のように好中球は、ヒトの血液サンプルから単離された。 </li><li>ジエチレントリアミン5酢酸（DTPA）は0.1mMを含むPBSで1 M DMPO *のストック溶液を作成します。 DMPOは、25から29の融点を有する°C、それがガラスバイアル（注に液体DMPO（密度〜1.02グラム/ mlの25℃）ピペッティングする方が便利ですので、：純粋なDMPOので、計量プラスチックバイアルを使用しないでください。 ）プラスチックと反応します 。凍結DMPOは、バイアル（：DMPOが分解可能性があるのでお湯を実行しないでください注）へとルーク温水を実行することによって溶融させることができる。 </li><li>それ以来、高純度DMPOを（&gt; 99％）を使用することが重要である市販のスピントラップの一部は、常磁性不純物を含んでおり、したがって、それは単独でだけDMPO溶液のEPRスペクトル（このケースでは10 mM）を実行することが不可欠です。それはバックグラウンド信号（ 図3Aを参照）は明らかではないことが重要です。 </li><li> DMSO中のホルボール-12 - ミリステート-13 - アセテート（PMA）のストック溶液（1 mg / ml）を準備します。 PBSで10μg/ mlの解決策を希釈することにより、アリコートを行います。 </li><li> 1。5ミリリットルエッペンドルフチューブには、添加の順序に従うことによって0.6ミリリットルの合計体積で解決策を準備します。PMNのmlあたり〜10 6細胞、D-グルコース（1 mg / ml）とアルブミン（1 mg / ml）を、10 mMのDMPOとPMA 0.2μg/ mlの。（注：PMAは、急進的な活性化因子であると最後に追加する必要があります）。 </li><li> EPRフラットセルに溶液を移します。 </li><li> EPR取得パラメータの条件は次のとおりです。マイクロ波周波数：9.8 GHzの、中央のフィールド：3486 G;変調振幅：0.5 G、掃引幅：100 G;レシーバゲイン：5×10 5;スキャンの総数：10;掃引時間：30秒、マイクロ波電力：20 mWで、そして一定時間：81 msです。付加体形成の定量化については、濃度対信号強度（または地域）の標準的なプロットは、そのようなテンポや-3 - カルボン酸-PROXYLとして安定したニトロキシドを用いて構築することができます。 </li></ol> DMPOの利用に関する重要な注意：フラットセルを使用することにより、一つの細胞密度を高め、それによって信号を増加させることができスピンアダクトの強さがO 2の半減期• - DMPOの付加物が短い（T 1/2〜1分）DMPO-OHに分解する。 DMPOは、t 1/2〜8とそれぞれ14分、と増加した付加物の安定性のために市販されているEMPO、BMPOまたはDEPMPOの同じ濃度を用いて置換することができます。 4。代表的な結果のFe 2からのEPRシグナルを示していない+ MGD又はFe 2の混合物は、CAIと+-MGDバックグラウンド信号があり、これらの試薬 ​​に由来しないことを示すラジカル+ MGD 図2Aおよび2BのFe 2を用いて行われたNOのスピン捕捉。のFe 2と反応してCAIのリリースNO刺激によりBAECスピン付加物を形成するために+-MGD、NO-のFe 2 +-MGDと、N = 12.66 Gの超微細分裂定数（HFSC）の値を持つ特徴的なトリプレット信号を示し、 G = 2.040のg因子。 （ 図2C）。 HFSC値は、NIEHS EPRソフトウェアデータベースのWebサイトからダウンロードすることができますWINSIMシミュレーションプログラムを用いて決定した。実験的なHFSCは、NO-のFe 2 +-MGD付加のためにN = 12.70 Gとg = 2.041 18の文献値と一致しています。同様に、BAEC上にSIN-1の効果は、 図2Dに示すように、eNOSのは、脱共役によるNO産生を低下させません。 検出- DMPOスピントラップは、O 2•使用されました。スピントラップは常磁性不純物から自由であることを確認し、図3Aに示すように、DMPOは、単独でシグナルを与えなかった。 図3Bは、唯一のDMPOとPMNのスペクトルであり、同様に、DMPOが発生しないことを示唆して検出可能なシグナルはありません酵素NADPHオキシダーゼの活性化を図3Cは、PMAによるPMNの刺激時に ​​観察されたEPR信号を示しています。この信号のHFSCの値は、N = 14.71 G、＆であると決定されたβ、-H = 11.40 G、γ-H = 1.25 G、およびN = 14.3 G、β-H = 11.7 Gとγ-H = DMPO-O 1.3 G 19の文献値と一致している2 H付加。 <img src="/files/ftp_upload/2810/2810fig1.jpg" alt="図1" /> 図1：EPRスピントラップを用いた好中球およびBAECからラジカルを検出するためのフローチャート。 （A）PMNはDMPOとPMAと混合し、得られた混合物は、データ取得のためのEPRフラットセルに転送されます。 （B）BAECは、プレート上に成長し、DPBSで洗浄した。スピントラップのFe（MGD）2 CAIと一緒に追加されました。溶液が十分に混合し、インキュベートした。混合物は、EPRのデータ取得のためのEPRフラットセルに移した。 <img src="/files/ftp_upload/2810/2810fig2.jpg" alt="図2" /> 図2 BからのNOのEPR検出AEC。のFe 2 + MGD + CAIの（）のFe 2スペクトラム+ MGDのみ（B）スペクトルのみ。 （C）のFe 2 NOトラップに起因するトリプレットスペクトルは+-MGD CAI-刺激した細胞を使用します。 （D）スペクトルは、0.5 mMの細胞のSIN-1処理によるNO産生の減少を示している。 <img src="/files/ftp_upload/2810/2810fig3.jpg" alt="図3" /> 図3活性化好中球からのDMPO-O 2 HのEPRを検出します。 10 mMのDMPOの（A）スペクトルのみ。 （B）の10 mM DMPOの存在下で単独でPMNのスペクトル。 （C）PMNのスペクトルは、10 mMのDMPOの存在下でPMAによって活性化。

<ol>
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Chem. 38, 258-265 (1995).</li><li>Snyrychova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+the+sensitivity+of+EPR+spin+trapping+in+biological+systems+by+cyclodextrins:+A+model+study+with+thylakoids+and+photosystem+II+particles.">Improvement of the sensitivity of EPR spin trapping in biological systems by cyclodextrins: A model study with thylakoids and photosystem II particles.</a> Free Radical Biol. Med. 48, 264-274 (2010).</li><li>Han, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipophilic+beta-cyclodextrin+cyclic-nitrone+conjugate:+Synthesis+and+spin+trapping+studies.">Lipophilic beta-cyclodextrin cyclic-nitrone conjugate: Synthesis and spin trapping studies.</a> J. Org. Chem. 74, 5369-5380 (2009).</li><li>Han, Y., Tuccio, B., Lauricella, R., Villamena, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+spin+trapping+properties+by+beta-cyclodextrin-cyclic+nitrone+conjugate.">Improved spin trapping properties by beta-cyclodextrin-cyclic nitrone conjugate.</a> J. Org. Chem. 73, 7108-7117 (2008).</li><li>Hardy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection,+characterization,+and+decay+kinetics+of+ROS+and+thiyl+adducts+of+mito-DEPMPO+spin+trap.">Detection, characterization, and decay kinetics of ROS and thiyl adducts of mito-DEPMPO spin trap.</a> Chem. Res. Toxicol. 20, 1053-1060 (2007).</li><li>Kim, S. -. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+reactivity+of+a+cyclic+nitrone-calix[4]pyrrole+conjugate+with+superoxide+radical+anion:+Theoretical+and+experimental+studies.">Fast reactivity of a cyclic nitrone-calix[4]pyrrole conjugate with superoxide radical anion: Theoretical and experimental studies.</a> J. Am. Chem. Soc. 132, 17157-17173 (2010).</li></ol>

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EPRスピントラップは、フリーラジカルを定量化し、識別するための生物医学アプリケーションの広い範囲で採用されている。スピントラッピングは、nMからこのように生物系のアプリケーションに適していμMの範囲の濃度でラジカルを検出することができる、非常に敏感である。常磁性の付加物の形成、NO-のFe 2 +-MGDは、EPR を経由して NOの検出の基礎となっています。のFe 2...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>フェノールフリーDMEM培地 高グルコース1X </td><td> GIBCO </td><td> 31053 </td><td></td></tr><tr><td> 0.25％トリプシン-EDTA </td><td> GIBCO </td><td> 25200 </td><td></td></tr><tr><td> L-グルタミン</td><td>フィッシャー·サイエンティフィック</td><td> BP379-100 </td><td></td></tr><tr><td> MEM非必須アミノ酸</td><td> GIBCO </td><td> 11140 </td><td></td></tr><tr><td>ウシ胎仔血清</td><td>アトランタバイオ</td><td> S11550 </td><td></td></tr><tr><td>内皮細胞増殖因子</td><td>ミリポア</td><td> 02から102 </td><td></td></tr><tr><td> CAI </td><td>エンツォ生命科学</td><td> A-23187 </td><td> DMSOに溶解</td></tr><tr><td> SIN-1 </td><td>エンツォ生命科学</td><td> BML-CN245-0020 </td><td></td></tr><tr><td> DMPO </td><td>同仁化学研究所</td><td> D048-10 </td><td></td></tr><tr><td>のFeSO 4·7H 2 O </td><td>シグマアルドリッチ</td><td> 215422-250G </td><td> CaとMgを含むPBSに溶解し</td></tr><tr><td> MGD </td><td>エンツォ生命科学</td><td> ALX-400-014-M050 </td><td>のCa 2 +およびMg 2を含むPBSに溶解+ </td></tr><tr><td> BAEC細胞</td><td>セルシステム</td><td> 2B2-C75 </td><td></td></tr><tr><td> DMSO </td><td>フィッシャー·サイエンティフィック</td><td> BP231-100 </td><td></td></tr><tr><td> DPBS </td><td>シグマアルドリッチ</td><td> D8537 </td><td></td></tr><tr><td>のCaCl 2とMgCl 2とDPBS </td><td>シグマldrich </td><td> D8662 </td><td></td></tr><tr><td>ホルボールミリステートアセテート（PMA） </td><td>シグマアルドリッチ</td><td> 79346-1mgの</td><td></td></tr></tbody></table>

detection of nitric oxide and superoxide radical anion by electron paramagnetic resonance spectroscopy from cells using spin traps

低濃度で反応性窒素/酸素種（ROS / RNS）は、シグナル伝達、細胞機能の調節に重要な役割を果たしていると、免疫応答が無秩序な濃度で細胞生存率を1、2に有害である。生きているシステムでは、ROSの生成を調節する内因性および栄養抗酸化防御機構と進化してきた一方で、ROSは、酸素の正常な代謝の副産物自然に連続的に製造されており、タンパク質の機能の喪失、DNAの切断、または脂質に生じる生体分子への酸化的損傷を引き起こす可能性があります過3、最終的に細胞傷害または死亡を4につながる酸化ストレスに。 スーパーオキシドアニオンラジカル（O 2• - ）は、ペルオキシナイトライトとヒドロキシルラジカルのような生物学的システムに存在することが知られている最も高度な酸化種のいくつかの主要な前駆体である。 O 2の生成• -私は、したがって、酸化的バーストの最初の兆候を通知し、TSの検出および/または生物学的システムの隔離が重要である。このデモでは、O 2• -多形核好中球（多形核白血球）から生成されました。ニコチンアミドアデニンジヌクレオチドリン酸（NADPH）オキシダーゼ5の活性化を介して-ホルボール-12 -ミリステート-13 -アセテート（PMA）と走化性刺激により、PMN。•O 2を生成します 。 3つのアイソフォームで提供一酸化窒素（NO）合成酵素は、誘導-、神経と血管内皮-NOS、またはiNOSの、nNOSのか、eNOSのは、それぞれ、番号6を生成しないようにNADPHを用いて、L-シトルリンにL-アルギニンの変換を触媒として。ここでは、内皮細胞からNOを生成しません。酸化的ストレス条件下で、例えばeNOSのは•O 2、NOを生産に切り替えることができます-ヘム7または補助因子の酸化によって引き起こされると考えられている脱共役と呼ばれるプロセスで、テトラヒドロビオプテリン（BH 4）8。 少数がありますしかし、生体系におけるフリーラジカルの検出のための信頼できる方法は特異性と感度によって制限されます。スピントラッピングは、一般的にフリーラジカルの同定に使用され、電子常磁性共鳴（EPR）分光法によって検出することができる永続的なスピン付加物を形成するスピントラップへのラジカルの付加反応を伴うされています。様々なラジカル付加物は、生成されるラジカルを識別し、ラジカルの産生9の性質と動態に関する豊富な情報を提供することができます使用することができる独特のスペクトルを示す。 環状ニトロン、5,5 -ジメチル-ピロリン-N-オキシド、DMPO 10、ホスホリル置換されDEPMPO 11、エステル置換EMPO 12 BMPO 13は 、広くスピントラップとして採用されている-後者のスピン付加-トラップは、O 2•のために長い半減期を示す。鉄（II）-N-メチル-D-グルカミンジチオカルバミン酸のFe（MGD）2 &lt;/ SUB&gt;は、一般的に付加体形成とスピン付加物14の高安定度の高い率のためにNOを捕捉するために使用されていません。

Watch this Scientific Journal Video about Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps at JoVE.com

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

電子常磁性共鳴（EPR）分光法はウシ大動脈内皮細胞および鉄（II）-N-メチル-D-グルカミンジチオ、Feを（MGD）を用いてヒト好中球からのスーパーオキシドラジカルアニオンから一酸化窒素を検出するために採用した<sub&gt; 2</sub&gt;と、それぞれ5,5 - ジメチル-1 - pyroroline-N-オキシド、DMPO、。

一酸化窒素とスピントラップを使用して、細胞からの電子常磁性共鳴分光法によるスーパーオキシドアニオンラジカルの検出

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

detection-nitric-oxide-superoxide-radical-anion-electron-paramagnetic

Research

JoVE Journal

Biology

18.9K ビュー.  The Ohio State University. 電子常磁性共鳴（EPR）分光法はウシ大動脈内皮細胞および鉄（II）-N-メチル-D-グルカミンジチオ、Feを（MGD）を用いてヒト好中球からのスーパーオキシドラジカルアニオンから一酸化窒素を検出するために採用した 2と、それぞれ5,5 - ジメチル-1 - pyroroline-N-オキシド、DMPO、。

ビデオ: Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

電子スピン共鳴酸素マッピングのためのライブ種のマイクロイメージング

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

生物学

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 ...

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

核磁気共鳴分光法を用いた環境メタボロミクスのための低密度プランクトンコミュニティからの代謝物の濃度

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

ライブの検出<em&gt;エシェリヒア·コリ</em&gt; O157：PMA-qPCRによりH7細胞

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

細胞内カルシウムの二光子イメージング<sup&gt; 2+</sup&gt;取扱い及び内皮における一酸化窒素産生および単離されたラット大動脈の平滑筋細胞

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

無傷の動脈の内皮層における一酸化窒素とスーパーオキシドのエン顔検出

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

の開発と特性評価<em&gt;インビトロ</em&gt;微小血管ネットワークおよび内皮の定量的測定の[Ca<sup&gt; 2+</sup&gt;]<sub&gt;私</sub&gt;と一酸化窒素の生産

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

高感度免疫組織化学を使用してシトシンの修飾された形態の検出

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

化学発光法を用いて、生物学的材料に、一酸化窒素経路に亜硝酸塩と硝酸塩、代謝物を測定します

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

Methods Article

一酸化窒素とスピントラップを使用して、細胞からの電子常磁性共鳴分光法によるスーパーオキシドアニオンラジカルの検出