This work was supported by the Swiss National Science Foundation (310030_141070/1), Swiss Heart Foundation, and National Center of Competence in Research (NCCR-Kidney.CH) Switzerland. Yu Yi is supported by the Chinese Scholarship Council.

動物の作業はフリブール、スイスの獣医事務所の倫理委員会によって承認されました。プロトコルは、私たちの施設で動物のケアと実験のガイドラインに従います。 単離された動脈のインキュベーションのためのセットアップの調製<ol><li> 37℃に加熱し、炭素ガスタンクから95％O 2および5％CO 2を通気することができる器官浴システムを構築します。 </li><li>以下の組成物の濃度に必要な量のクレブス-リンガー重炭酸緩衝液を準備します（118のNaCl、4.7のKCl、2.5mMのCaCl 2を、1.2 mMの硫酸マグネシウム、1.2 mMのKH 2 PO 4、25mMのNaHCO 3を、0.026 mMのEDTA、および11.1 mMのD-グルコース）。 </li><li>氷上ストック緩衝を維持し、95％O 2および5％CO 2でバッファを通気。 </li><li>オーガンバスシステム上のスイッチ、37℃に温度を設定し、各チャンバーにすぐに使用できるバッファの5ミリリットルを追加95％O 2および5％CO 2でバッファを通気保ちます。 </li><li> 30分間クレブス - リンガー緩衝液で1回チャンバーを洗浄します。 </li><li>各チャンバーに5ミリリットルクレブス - リンガー緩衝液を添加し、蒸発を避けるために、チャンバーをカバーしています。 </li></ol>マウス大動脈の2の単離<ol><li>マウスを犠牲にする150 10mg / kgの濃度で腹腔内の0.9％NaClに溶解されたペントバルビタールを注入します。 </li><li>手術用ボードに無気力にマウスを置きます。 </li><li>消毒や水分の目的のために70％エタノールで胸部の毛スプレー。 </li><li>心臓や肺を露出させ、肺を除去するために、リブの周りに切断してオープン胸腔。 </li><li>ティッシュペーパーで軽く胸腔内の血液を除去します。 </li><li>ピンセットでそっと心をつかみ、手術用ハサミで大動脈と背骨の間の血管周囲脂肪組織を切断することにより、胸部大動脈を切り離します。 </li><li>すぐにwholを浸します氷冷（4℃）クレブス - リンガー緩衝の電子組織。 </li><li>解剖顕微鏡下で心臓や大動脈弓を削除し、バッファに胸部大動脈セグメントを保ちます。 </li><li>手術用ハサミやピンセットで顕微鏡下で血管周囲の組織を接着するから無料大動脈を解剖。 </li><li>鉗子で大動脈の端縁をつかみ、ゆっくり26 G×1 / 2&quot;シリンジでクレブス - リンガーバッファをフラッシュすることにより、血管の内腔に血液を洗い流します。 </li><li> 3ミリメートル長いセグメントに洗浄大動脈輪をカットします。 注：内皮層に損傷を与えないために、この段階で注意を払ってください。 </li></ol> 3. DHEとDAF-2DA染色<ol><li> 37℃のクレブス-リンガー緩衝液で満たされた臓器槽チャンバに鉗子で洗浄大動脈セグメントを転送するには、95％O 2および5％CO 2を通気。 鉗子で大動脈環のタッチだけ外膜側をaとbを固定しないでください注：lood船。 </li><li> 30分間器官槽チャンバ内の動脈を平衡化します。 </li><li>最終濃度1μMの器官槽にアセチルコリン（AChの）を追加し、10分間動脈をインキュベートします。 </li><li> 1.5ミリリットルマイクロチューブに予め温めたクレブス - リンガー緩衝液で（千×ストックから希釈した各色素の5μM）DHE / DAF-2DA溶液1mlを準備します。光暴露を避けるためにアルミホイルでマイクロチューブを包みます。 </li><li>刺激後、37℃でマイクロ遠心チューブにDHE / DAF-2DA溶液に器官浴から大動脈環を転送、95％O 2および5％CO 2を通気し、30分間大動脈環をインキュベートします。 注：このステップから、暗闇の中で常に大動脈を保ちます。 </li><li>洗浄のためにクレブス - リンガー緩衝液で新しいマイクロチューブに大動脈輪を移し、1分以内に洗浄を3回繰り返します。 </li><li> 30固定のために4％パラホルムアルデヒド溶液に大動脈環を転送します分。 </li><li>準備1ミリリットル4 &#39;、リン酸緩衝生理食塩水（PBS）を1.5 mlのマイクロ遠心チューブ内のバッファを持つ（千×ストックから希釈した300 nMの、）6-ジアミジノ-2-フェニルインドール（DAPI）ソリューション。 3分間の大動脈を対比染色。 </li><li> 1分間の洗浄のためにPBS緩衝液で新しいマイクロチューブに大動脈を転送します。洗浄を3回繰り返します。 注：大動脈輪をクランプまたは内皮層を傷つけないよう注意してください。 </li></ol> 4. アンフェイスマウント<ol><li>スライドにマウンティング培地のドロップを追加します。 </li><li>ロールアップ大動脈を平らにし、スライドガラス上に下向きに内皮とマウンティング媒体に顔エンそれをマウントし、顕微鏡下で顕微はさみで縦方向に大動脈リングをカット。前後に大動脈を移動しないでください。 </li><li>カバースリップとスライドを覆い、マニキュアとスライドを封印。 </li><li>光保護下に空気乾燥した後、悲惨な画像化するためのスライドを使用CTLY時間以内または今後数日間、-20℃で保管してください。 </li></ol> 5.共焦点顕微鏡イメージング<ol><li>蛍光シグナルを検出するために、共焦点顕微鏡を使用してください。マシンに切り替えて、このような倍率、スキャン速度、分解能、Zスタックなどの画像設定を最適化。 <ol><li>このプロトコルでは、200 Hzの走査速度、1024×1024ピクセルの解像度および0.25μmでのZ-ステップサイズを使用します。一方、514 nmでのDHEから蛍光を励起し、605 nmの発光で検出し、488nmのアルゴンレーザーをDAF-2DAから蛍光を励起し、515 nmで検出します。 </li></ol></li><li> DAPI信号が紫外線を透明になるまで、サンプルに焦点を当てる10X倍率を使用し、その後、40倍の倍率に目標を調整します。 </li><li>コントロールパネル上のZ位置を調整することにより、内皮層の範囲を定義します。 DAPI染色された核の信号は、内皮層における楕円形や円形のドットです。 </li><li>上からスキャン内皮信号と記録画像の全厚を通る試料の（内腔の境界線上に内皮層）。各サンプルを走査するための少なくとも3つの異なるフィールドを選択してください。 </li></ol>画像の6.分析<ol><li>共焦点顕微鏡によってスキャンしたデータを開くためにソフトウェアを使用してください。分析のためのフィールドごとに3つの連続した​​画像を選択し、サンプルあたり少なくとも3つの異なるフィールドを評価します。選択した画像をエクスポートし、JPEGファイルとして保存します。 </li><li>画像処理ソフトでDAF-2DA、DH​​E及びDAPI染色から画像を定量化します。 DAPI染色からの画像は、DAPI陽性の核の数をカウントする「プラグイン」→「分析」→「セルカウンター &#39;を選択します。 <ol><li> DAF-2DAとDHE染色からの画像では、信号の強度を分析し、相対シグナル強度として「平均」の値を取る→「メジャー &#39;&#39;を分析する」を選択します。 DAF-2DAの比にとして存在し、結果DAPI陽性核またはDAPIに対するDHEの比率。各サンプルについて、すべての画像およびフィールドの平均値をとります。 </li></ol></li><li>倍の変化を取得するには、対照群の平均値で各サンプルの結果を割ります。統計分析は、ダネットまたはボンフェローニ事後検定を用いて不対スチューデントt検定またはANOVAを用いて行きました。平均±SEMとしてデータを与えます。 p &lt;0.05の14で有意で、平均値の差を考慮してください。 </li></ol>

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No conflicts of interest declared.

NOまたはO 2の検出は、.-蛍光色素で頻繁に組織の凍結切片23にも培養内皮細胞における多くの研究で使用されました。ここでは、 顔のNOの検出とO 2 .-、比較的単純で、かつ直感的に有効であるそれぞれDAF-2DAとDHE、と内皮層におけるレベル途中 、 すなわち 、無傷の生きた血管、この方法を拡張しました。血管の凍結切片での方法と?...

血管内皮細胞は、血管作動性因子1を放出することによって血管の機能的および構造的完全性を維持します。これらの要因の中では、内皮NOシンターゼ（eNOSの）を介して、L-アルギニンから生成された内皮由来一酸化窒素（NO）は、心臓血管生理学2の中で最も重要かつ最もよく特性要因です。 NOは、平滑筋の弛緩を引き起こし、細胞増殖を阻害し、したがって血管疾患の発達3に対する保護、内皮下の空間に血小板凝集、および炎症性細胞接着および浸潤を抑制する。加齢、高血圧、糖尿病、高脂血症などを含む多くの生理学的および病理学的条件下では、することを特徴と内皮機能障害は、NO生物学的利用能を低下させず、O 2を増加させた .-生産が存在し、アテローム性動脈硬化症2の病因を促進します。近年の研究では、eNOSのの脱共役であることを実証しています様々な上記条件1,4の下でのeNOSの酵素は、O 2を生成する内皮機能不全、.-代わりにNOのための重要なメカニズム。したがって、内皮機能の分析は、特に、NO産生を内皮ず、O 2 .-世代は、心血管疾患や合併症に関する実験的研究のために極めて重要です。 生物学的サンプル中のNO産生を分析せず、測定するために開発されてきた多数の方法論的アプローチがあります。容易にNO 2に酸化されるNOの極めて不安定な性質のために-及びNO 3 - 3〜6秒の半減期で、直接測定しないことは困難です。したがって、NO 2の決意- / NO 3 -流体サンプル中の細胞または組織5から放出されたNOの指標としました。手順は比較的容易であるが、この方法は、しかしながら、EA/ NO 3 - -溶液中に含まれるsily安定したNO 2の高いバックグラウンドによって影響を受けます。 NOは、環状グアノシン一リン酸（cGMP）6を生成するために可溶性グアニル酸シクラーゼを刺激するため、細胞のcGMPレベルはまた、NO放出7を推定しないと判定されました。繰り返しますが、これは間接的な推定値であり、そのようなC型ナトリウム利尿ペプチド（CNP）のようないくつかの内皮由来の要因も、粒子状グアニル酸シクラーゼ8の活性化を介してcGMPレベルを高める可能性があるので、特定できない場合があります。 NOは、副生成物9、L-シトルリン生産の測定は、したがって、NO産生を推定しないように間接的な方法として使用されているように、L-シトルリンの生成とL-アルギニンから生成されます。この方法の主な欠点は、それが放射性であり、それは、生物活性NOレベルを測定しないことがあり、O 2によってNOが急速に不活性化することができた解放以来.-。また、L-シトルリンは、L-ためにリサイクルすることができます10アルギニン 。このような化学発光検出11、電子スピン共鳴12、または電気化学ポルフィリンNOセンサ13などの他の化学的方法は、いくつかの研究者によって使用されています。これらの方法は、通常、検出手順の動作が容易ではなく、特殊な装置を必要とします。これは、多くの研究は、内皮機能を評価し、間接的に内皮由来NO媒介血管弛緩を測定するために、内皮の有無にかかわらず、単離された血管と器官槽実験を適用することを言及することもあります。それは生理学的状況に主に近いですが、厳密に言っているが、NO機能を測定しません。しかし、この方法では、それはむしろeNOSの機能の正味の影響を反映し、一般的に内皮媒介血管運動応答、他の内皮由来弛緩の生産を評価します要因と内皮由来契約要因、O 2 .-の生産、また、平滑筋細胞の応答これらの要因に。 eNOSの機能またはNO産生の具体的な分析は、通常3を必要とされています。 我々を含む多くの研究グループは、近年NO 14〜19の細胞内産生を検出する蛍光色素法を使用しています。この方法では、細胞透過性の蛍光標識ジアミノ-2アセテート（DAF-2DA）は、in vitroまたはex vivo での細胞および生体組織中の遊離NOおよびNOS機能を測定しました。原理は、生きた細胞内で、DAF-2DAは、その後、NOと反応させることにより、DAF-2トリアゾール（DAF-2T）を蛍光性に変換した非蛍光4,5-ジアミノ（DAF-2）に細胞内エステラーゼにより脱アセチル化されていることです。 DAF-2Tからの蛍光は、蛍光顕微鏡または蛍光共焦点顕微鏡14で観察することができます。細胞内の蛍光強度は、したがって、細胞の細胞内NO産生または無傷の血液vesse内の内皮を反映しますリットル。このようなジヒドロ（DHE）のような特定の蛍光染料と組み合わせることで、1は、同時に細胞または血管14に細胞内NOとO 2 .-世代を評価することができます。同様に、DHEはまた、O 2 .-、細胞内で酸化された細胞透過性化合物、および酸化物であるその後、蛍光顕微鏡や蛍光共焦点顕微鏡により定量的に検出可能な鮮やかな赤色を発光する核酸とインターカレー。 DHEは、O 2を検出するための非常に特異的な色素である.-生物学的試料から、それは本質的に、スーパーオキシドラジカルを検出すると、細胞によって十分に保持され、さらに穏やかな固定20を許容することができます。この蛍光色素法の利点の一つは、それが検出され、直接に生体血管の無傷内皮層の面エン NOおよび/ ​​またはO 2を .-可視化することです。 本論文では、我々は、この蛍光色素NOを検出する方法とO 2 .-私たちはNOとO 2の顔検出専用のために適合している.-高脂肪食（HFD）の給電により誘導される肥満モデルマウスの無傷の大動脈でについて説明します。我々は、このメソッドが正常にかつ確実にNOとO 2 .-レベルを測定し、肥満で新たに単離した無傷のマウスの大動脈の内皮層中のeNOS（DYS）機能を評価することができることを実証します。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dihydroethidium (DHE)</td>
			<td>Invitrogen</td>
			<td>D 1168</td>
			<td>dissolve with DMSO to 5 mmol/L as 1000X stock, stored at -20&#176;C</td>
		</tr>
		<tr>
			<td>Diaminofluorescein-2 Diacetate (DAF-2 DA)</td>
			<td>Calbiochem</td>
			<td>251505</td>
			<td>dissolve with DMSO to 5 mmol/L as 1000X stock,stored at -20&#176;C</td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Invitrogen</td>
			<td>D 1306</td>
			<td>dissolve with water to 300 &#181;mol/L as 1000X stock,stored at 4&#176;C</td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td>Vector labor. (reactolab)</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Name (N o-nitro-l-argininemethylester.HCl)</td>
			<td>Sigma-aldrich</td>
			<td>A5751</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Sigma-aldrich</td>
			<td>P3636</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-Myograph System&#160;</td>
			<td>Danish Myo Technology A/S</td>
			<td>Model 610M</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope&#160;</td>
			<td>Leica&#160;</td>
			<td>DM6000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Image processing software</td>
			<td>National Institute of Health(NIH)</td>
			<td>Image J&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors&#160;</td>
			<td>S&#38;T AG</td>
			<td>SDC-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical scissors&#160;</td>
			<td>F.S.T</td>
			<td>15000-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>S&#38;T AG</td>
			<td>JF-5</td>
			<td></td>
		</tr>
		<tr>
			<td>26G&#215;1/2&#698; syringe</td>
			<td>Becton Dickinson</td>
			<td>305501</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip round diam15mm</td>
			<td>vwr</td>
			<td>631-1579</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips 1 mL</td>
			<td>vwr</td>
			<td>RFL-1200c&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips 200 uL</td>
			<td>vwr&#160;</td>
			<td>613.0659</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes 1.5 mL</td>
			<td>Eppendorf&#160;</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetylcholine chloride</td>
			<td>Sigma-aldrich</td>
			<td>A-6625</td>
			<td></td>
		</tr>
	</tbody>
</table>

en face detection of nitric oxide and superoxide in endothelial layer of intact arteries

内皮NOシンターゼ（eNOSの）から製造された内皮由来一酸化窒素（NO）は、心臓血管生理学の中で最も重要な血管保護分子の一つです。そのようなのeNOSの脱共役としての機能不全のeNOSは、スーパーオキシドアニオンにおけるNO生物学的利用能および増加の減少につながる（O 2 .-）の生産、ひいては心血管疾患を促進します。したがって、内皮細胞におけるNOとO 2 .-レベルの適切な測定は、心血管疾患や合併症の研究のために極めて重要です。なぜならNO及びO 2 .-の極めて不安定な性質のため、測定及びO 2 .-直接血管内のが困難です。多くの方法は、NOとO 2 .-産生を測定するために開発されています。しかしながら、いずれかの非感受性、または非特異的な、または技術的に要求され、特別な装置を必要とします。ここでは、適応を説明します顔を同時に検出し、細胞内のNOとO 2の可視化エン .-誘発性肥満マウスモデルの無傷の大動脈で、それぞれ、細胞透過性ジアミノ-2ジアセテート（DAF-2DA）およびジヒドロ（DHE）を使用するための蛍光色素法の高脂肪食負荷によって。我々は、細胞内のNOを減少し、コントロールリーンマウスと比較して、O 2 .-肥満マウスの新たに単離された無傷の大動脈内のレベルを高め実証できました。我々は、この方法は、NOとO 2の直接検出と可視化.-無傷の血管のための簡単な技術であり、広く（生理）病理学的条件下での内皮（DYS）関数の調査のために適用することができることを実証しています。

肥満は虚血性冠動脈性心疾患の重要な危険因子であり、関連するとNOの生物学的利用能、アテローム性動脈硬化性血管疾患21の特徴を内皮ない減少します。 eNOSの-脱共役内 ​​皮22老化を含む多くの生理学的および病理学的条件下での機能不全、アテローム性動脈硬化症、及び肥満14の重要なメカニズムであることが示されています。そこで、?...

Watch this Scientific Journal Video about En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries at JoVE.com

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

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

この記事では、 顔を同時に検出し、細胞内の一酸化窒素（NO）とスーパーオキシドアニオン（O 2の可視化エンための蛍光色素ジアミノ-2ジアセテート（DAF-2DA）およびジヒドロ（DHE）を使用して適応が比較的容易な方法を記載しています.- ）は、それぞれ、肥満マウスモデルの新たに単離された無傷の大動脈です。

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

en-face-detection-nitric-oxide-superoxide-endothelial-layer-intact

Research

JoVE Journal

Biology

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

9.5K ビュー.  University of Fribourg. この記事では、 顔を同時に検出し、細胞内の一酸化窒素（NO）とスーパーオキシドアニオン（O 2の可視化エンための蛍光色素ジアミノ-2ジアセテート（DAF-2DA）およびジヒドロ（DHE）を使用して適応が比較的容易な方法を記載しています.- ）は、それぞれ、肥満マウスモデルの新たに単離された無傷の大動脈です。 

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

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

ELISAによるネイティブ無傷ヌクレオソーム上の翻訳後修飾の検出

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

生物学

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

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

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 ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

マウス抵抗動脈から毛細血管内皮の管の隔離

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

シングルミトコンドリアスーパーオキシド無傷ハートや点滅の共焦点イメージング<em&gt;インビボ</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Analysis of Oxidative Stress in Zebrafish Embryos

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

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

ゼブラフィッシュ胚における酸化ストレスの分析

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

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 ...

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 ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

「せん断リング」の組み立てと応用：小説内皮軌道、単方向および定期的な流動のためのモデルとせん断応力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

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 ...

En Face Preparation of Mouse Blood Vessels

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the three-dimensional tissue morphology is from such sections. In addition, the sections of tissues examined may not contain the region within the tissue that is necessary for the purpose of the ongoing study. This latter limitation hinders histopathological studies of blood vessels since vascular lesions develop in a focalized manner. This requires a method that enables us to survey a wide area of the blood vessel wall, from its surface to deeper regions. A whole mount en face preparation of blood vessels fulfills this requirement. In this article, we will demonstrate how to make en face preparations of the mouse aorta and carotid artery and to immunofluorescently stain them for confocal microscopy and other types of fluorescence-based imaging.

En Face Preparation of Mouse Blood Vessels

A procedure for making en face preparations of the mouse carotid artery and aorta is described. Such preparations, when immunofluorescently stained with specific antibodies, enable us to study localization of proteins and identification of cell types within the entire vascular wall by confocal microscopy.

マウスの血管の顔面準備

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the ...

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

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