我々は、イメージング研究のヘルプは博士ウィリアムCashdollar、およびウィスコンシン州の血液研究所のノースウェスタン·ミューチュアル財団イメージングセンターに感謝します。また、この原稿で、有用な議論の重要な読書のための博士ダリアIlatovskayaに感謝します。本研究は、（AS）に健康補助金HL108880と（AMG）はDP2-OD008396の国立研究所によってサポートされていました。

以下に記載の実験手順は、ウィスコンシン医科大学の施設内動物管理使用委員会（IACUC）によって承認され、実験動物の管理と使用に関する健康ガイドの国立研究所に応じてありました。 ラット大動脈の1の単離<ol><li>イソフルラン（5％誘導、1.5から2.5パーセントのメンテナンス）または別のIACUC承認の方法でラットを麻酔。 </li><li>仰臥位でラットを配置します。腹部の臓器を露出させるために、皮膚と皮下組織を通って腹部の小さな腹側正中切開を行います。ガーゼパッドを使用して、穏やかに、図1に示すように、大動脈と大静脈を露出するために腸を偏向させます。 </li><li>簡単に言えば、結合組織からと大静脈から大動脈を解剖鈍いです。縫合糸を使用して、腎臓にできるだけ近い大動脈を縛ります。大動脈2センチ、15ミリリットルの円錐TUに配置 - 約1を分析氷の上で：;（140のNaCl、1.2のMgCl 2、2のCaCl 2、4.5のKCl、6グルコース、10 mMのHEPESでPSS）は、通常の生理食塩液を含むこと。 </li><li>大動脈が分離されると、承認されたIACUCプロトコルに従って動物を安楽死させます。すべての非生存手続きの完了時に動物の人道的な終焉を確実にするために開胸誘導気胸により深く麻酔をかけた動物を安楽死させる。3 </li><li>解剖顕微鏡を使用して、先の細いピンセット、およびmicroscissorsは大動脈のオフ脂肪および結合組織を解剖。大動脈がきれいになったら、縦に大動脈を切断し、後で氷上PSSに配置します。 </li></ol> 2.色素添加およびインキュベーション<ol><li>アルミ箔で覆われている個々の500μlのチューブに、DMSOベースのソリューションで提供されている1 mMののFluo-4 AM色素のピペット7μlの。時間をかけて、色素の性質を維持するために-20℃の冷凍庫に保管してください。注：これらは非常に分注しlutionsは、少なくとも6ヶ月間安定であるべきです。 </li><li>使用前に、色素溶液を開く前に、室温まで昇温することができます。使用直前に作業溶液を調製します。後で使用するために希釈された試薬を保管しないでください。 </li><li>何のカルシウムを含有しないPSSの450 ULにDMSO溶液に溶解した既製の1mMのFluo-4 AM色素の7μlを添加します。アセトキシメチル（AM）エステルを分散させる助け、カルシウム色素の負荷を改善するために、ローディングカクテルに非DMSOベースの10％プルロニック酸溶液40μlを追加します。 </li><li> 1時間（2.3で説明したように）、ローディングカクテルを含む500μlのチューブに解剖した大動脈を置きます。 RTで回転シェーカー上箔と場所ですべてのチューブをカバーしています。 </li><li>血管系におけるNO産生を監視しない場合は、DAF-FMジアセテート染料を使用しています。 450μlのPSS、40μlのプルロニック酸および10mM DAF-FMジアセテートの5μLを含有する溶液中に大動脈をインキュベートします。 （2.3で説明）のカルシウムのインキュベーションプロトコルと同様に、大動脈を配置室温で次の30分間、続いて4℃で30分間回転シェーカー上に箔と場所でカバーNO染料溶液を含む500μlのチューブです。 </li><li> NO産生が測定されていない場合、 図4（b）に示すように、対照としてNO産生を遮断しないように、内皮のNO合成酵素阻害薬、L-NAMEを使用します。その手順については、（室温で）インキュベーションの最後の30分間（2.5で説明したように）カクテル100 nmのL-NAMEに追加します。 </li></ol> 3.レーザー走査2光子顕微鏡のプロトコル<ol><li>細胞外色素を除去し、クリーンPSSで3回 - 1時間のインキュベーションの後、大動脈2を洗います。 </li><li>通常のPSSまたはCa 2+を含まない緩衝液（実験プロトコルに依存します）のいずれかを含むシリコーン被覆皿に大動脈を転送します。ピンダウンμ状のピンを使用してシリコーン（皿開口部に露出され、すなわち、内皮ルーメン）と接触している外膜を有するシリコーンコーティング上に大動脈。あるいは、スライスアンカーを使用グリッドまたは接着剤は、組織を固定します。 注：ストレスと可能な機械的なアーティファクト、伝達を低減およびCa 2+フリーソリューションを大動脈を修正し、5待つに-実験開始の10分前。 </li><li>薬物の自動または手動のアプリケーションのために、または細胞外溶液を変更するためのシリコーン被覆プレートに蠕動ポンプまたはシリンジを接続します。 </li><li>直立二光子顕微鏡のノーズピースの下に皿を置き、試料の上に25×（NA 1.05と作動距離2mm）を水浸対物レンズを取り付け、位置決めします。注：この特定のレンズが使用できない場合は、通常の目的と比較して飛躍的に信号の解像度を向上させることができるので、特に2光子イメージングのために製造され対物レンズを使用しています。 </li><li>ソフトウェアを使用して、2つの光子レーザーをアクティブにします（レーザーは、ポンプとオンし始めます）。チューニング820 nmのレーザー励起。 </li><li>エピ蛍光を用いて、大動脈を探し、上の目的を集中粗調整を使用して、内皮表面。 </li><li>励起レーザは、モードロックされ、非デスキャン検出器が係合されると予想される発光スペクトルに適切な放射フィルタがインストールされていることを確認して、2光子レーザ走査顕微鏡モードに切り替えます。注：ここでは、FV10-MRL / R（540 nmの495）を使用しました。 </li><li>約5％のレーザー出力を使用して、生のスキャンを開始し、微細血管内皮細胞を可視化するために、目的の焦点を合わせます。 注：通常のPSSでインキュベートしたときに内皮細胞は強い蛍光シグナルを示すであろう。容器はカルシウムを含まない緩衝液中でインキュベートされたとき、これは著しく弱くなります。内皮細胞は、開いた大動脈の上に存在し、平滑筋細胞は、単に内皮細胞（ 図2参照 ）の下に見られます。血管はいずれも平滑であってもあるので、平滑筋細胞および内皮細胞の両方が存在している場所があります。&lt;/ LI&gt; <li>セルがフォーカスに入ると、顕微鏡ソフトウェアプログラムは、高速スキャンモードで連続画像（ラスタースキャン、1.1秒のフレームコレクションの頻度で512×512ピクセルのウィンドウ）を収集します。フルオ4 AM蛍光と並行して、血管収縮の変化を検出し、より良い実験条件を可視化するために光画像を送信し収集します。 </li><li>ベースライン信号画像のコレクションの後、外液中のCa 2+イオン濃度を変更したり、ゆっくりと撮影しながら蠕動ポンプを使用して、薬理学的な刺激剤を適用します。負荷細胞内の蛍光シグナルの増加を観察します。 注：内皮細胞は、フローの変化にこのようにして、それがのCa 2+またはNOシグナル伝達の薬理学的活性剤を塗布する前に、低い層アプリケーションや車両試験を使用することをお勧めし応答します。のFluo-4（のFluo-4 Iの解離定数がロードされた血管内nMの濃度値を、細胞内カルシウムを再計算しますS 345 nM）を、蛍光強度は、ベースライン時およびイオノマイシンとのMnCl 2を添加した後に記録することができた。4 </li></ol> 4.画像処理と計算<ol><li>フィジーの画像処理パッケージをダウンロードし、インストールします。 </li><li>フィジーの画像ファイルを開きます。プロンプトが表示されたら、ファイルをインポートすると、チャンネル（透過光と蛍光信号）を分割します。データ分析のための唯一の蛍光シグナルを使用してください。 </li><li>フィジープログラム内で、分析タブをクリックし、[ツール]オプションまでスクロールダウンします。ツールのオプションでは、ROIのマネージャーを言うタブを見つけ、それをクリックしてください。新しいウィンドウが表示されます。 </li><li>この後、分析]タブで設定された測定値をクリックしてください。関心のある特定の測定値を選択します。カルシウムトランジェント測定では、平均グレー値のオプションを使用します。 </li><li>円形トレースツールでは、関心領域（ROI）を追跡し、すべてのRのためのROIマネージャ画面で「追加」をクリックして始めますOI。一度興味のあるすべてのセルは、「より」タブの下にマルチメジャーを選択し、ROIマネージャウィンドウで、トレースされています。スプレッドシートにこれらの測定の全てを直接測定値を保存するか、コピー＆ペーストするかを覚えておいてください。 </li><li>フィジーから* .txtファイルやExcelスプレッドシートを開き、新しい原点ワークシートに番号を転送します。注：または、さらなる分析のためにシグマプロットやプリズムのようなプログラムを使用します。 </li><li>オリジンプログラム内では、すべての過渡応答の概要を観察するためにプロット→線+シンボルメニューを使用します。応答しない細胞の選択を解除します。 </li><li>時間スケール（X軸）へのトレース数を再計算し、列→列値の設定を使用し、（我々の場合1.1sで）画像収集頻度へのトレース番号の列を掛けます。 </li><li>平均（Y軸）および標準エラーのトレースを計算するために選択したすべての記録のための行の解析→統計を使用してください。細胞プロット→線+シンボルの現在のグループのための最終的なグラフをプロットします。 </li><li> MEA以下のようにn個のカルシウム過渡計算が記載されています。 <ol><li>オリジンプログラム内の総カルシウム放出のために、グラフをクリックして、次に統合→メニュー分析→微積分を使用しています。曲線下面積の全積分は、結果ログに表示されます。 </li><li>一過性の動態については、グラフ上でクリックし、メニューを使用します。分析→非線形曲線フィット→[データセット（最大値から過渡応答の最後の範囲でX軸を選択します）。完了→指数関数的減衰1→関数選択→フィッティングを開始します。 注：指数フィッティングがグラフウィンドウに表示され、ログイン結果。得られたデータ（値）をリリースし、カルシウムの総量を表し、チャンネルの動力学は、過渡応答を媒介しました。 </li></ol></li></ol>

<ol>
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著者らは、開示することは何もありません。

実験の概要。より良い血管生理学にカルシウムの寄与を理解し、NOに、新規な方法を測定するために開発されたの[Ca 2+] iと内の平滑筋および単離された無傷の大動脈の内皮細胞NO。 1）機械的分離と準備（酵素消化ない）容器の：一緒に、このプロトコルは、これらの重要なステップで構成されています。これは最適な生理学的記録を得ることが可能な限り健康で無傷?...

カルシウムは、内皮細胞および平滑筋細胞などの血管細胞内の基本的なセカンドメッセンジャーです。これは、血管収縮のための主要な刺激であり、内皮内のNO発生を介してその効果を含め、血管拡張において重要な役割を果たしています。これにより、撮像技術の制限のために、それは、無傷の血管内カルシウム処理を観察することは事実上不可能でした。 2光子イメージングシステムと新しいカルシウムの作成またはNO標識色素の開発は、カルシウム動態及び血管系内のNO産生を理解し始めるために十分な深さと解像度でイメージすることができます。 二光子顕微鏡は、最近ので、深く低いバックグラウンド蛍光と高い信号感度で組織に浸透するその優れた能力の組織、器官、さらには全体の動物試験に適用されている。1,2イルミネータで2光子励起の狭いスペクトルイオン焦点と非デスキャン検出器の使用は、2光子顕微鏡は、従来の共焦点顕微鏡よりも優れている理由です。共焦点顕微鏡は、自動蛍光および共焦点ピンホールへのピンボケ光の散乱に起因する必要な組織深さで高品質の画像を生成することはできません。したがって、我々は、[Ca 2+] i のシグナルを測定するために2つの光子顕微鏡を使用する方法と、高解像度、低信号対雑音比を有する無傷の、個々の血管細胞におけるNO産生を開発しました。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DAF-FM</td>
			<td>Life Technologies</td>
			<td>D-23842</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo 4 AM</td>
			<td>Life Technologies</td>
			<td>F14217</td>
			<td>500&#181;l in DMSO</td>
		</tr>
		<tr>
			<td>Pluronic F-68 solution</td>
			<td>Sigma-Aldrich</td>
			<td>P5556</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Name</td>
			<td>Tocris</td>
			<td>0665</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus upright microscope</td>
			<td>Olympus</td>
			<td>Fluoview FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>MaiTai HP DeepSee-OL&#160;</td>
			<td>Spectra Physics</td>
			<td></td>
			<td>Ti:sapphire laser 690nm &#8212; 1040nm</td>
		</tr>
		<tr>
			<td>Filters</td>
			<td>Olympus</td>
			<td>FV10-MRL/R&#160;</td>
			<td>495 to 540 nm&#160;</td>
		</tr>
		<tr>
			<td>25&#215; water-immersion objective lens</td>
			<td>Olympus</td>
			<td>XLPL25XWMP</td>
			<td>N.A. 1.05 and working distance 2 mm</td>
		</tr>
		<tr>
			<td>Slice Anchor grid&#160;</td>
			<td>Warner Instrument&#160;</td>
			<td>SHD-27LH</td>
			<td></td>
		</tr>
		<tr>
			<td>Other basic reagents&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

two-photon imaging of intracellular ca2+ handling and nitric oxide production in endothelial and smooth muscle cells of an isolated rat aorta

カルシウムは、血管組織の多くの生理学的プロセスの非常に重要な調節因子です。ほとんどの内皮および平滑筋の機能は非常に細胞内カルシウム（[Ca 2+] i） の一酸化窒素（NO）の変化に依存します。の[Ca 2+] i の 、NOおよび下流分子は血管収縮薬および血管拡張薬に反応して血管によってどのように処理されるかを理解するために、我々はカルシウム標識（またはNO標識）を適用する新規な技術を開発した2光子顕微鏡を有する色素単離された血管におけるカルシウム処理（またはNO産生）を測定しました。ここで説明した内皮細胞または平滑筋細胞内にラット、ラベルカルシウムまたはNOから大動脈を分離する方法を示す詳細なステップバイステップの手順であり、画像のカルシウムトランジェント（またはNO産生）生理次の2光子顕微鏡を用いて、または薬理学的刺激。この方法を使用する利点は、複数倍である：1）それは、同時にすることが可能ですLY異なる刺激に応答して、両方の内皮細胞および平滑筋細胞におけるカルシウムトランジェントを測定します。 2）それは、画像の内皮細胞とそれらの天然の環境における平滑筋細胞のいずれかを可能にします。 3）この方法は、細胞内カルシウムまたはNO変化に非常に敏感であり、正確な測定のための高解像度の画像を生成します。 4）記載の手法は、活性酸素種のような他の分子の測定に適用することができます。要するに、2光子レーザ発光顕微鏡の応用カルシウムトランジェントを監視し、単離された血管の内皮細胞および平滑筋細胞におけるNO産生は、高品質の定量的データを提供しておらず、血管機能を調節するメカニズムの理解を推進してきました。

正確に血管の生理学（血管拡張および血管収縮）のカルシウムの寄与を評価するために、プロトコルは、単離された無傷の大動脈の両方の内皮細胞および平滑筋細胞にカルシウム色素をロードするように設計されました。 図1に示されている設定の一般的な実験では、単離およびイメージングの前に容器の製造のための基本戦略を示しています。簡単に言うと、ラッ?...

Watch this Scientific Journal Video about Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta at JoVE.com

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 ...

two-photon-imaging-intracellular-ca2-handling-nitric-oxide-production

Research

JoVE Journal

Biology

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

11.2K ビュー.  Medical College of Wisconsin. 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. 

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

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

ライブゼブラフィッシュの胚における二光子軸索切断とタイムラプス共焦点イメージング

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

生物学

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

焦点のCa 2 +</sup平滑筋の>過渡検出

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

ヒト臍帯動脈平滑筋細胞の単離（HUASMC）

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

薬理研究に単離された組織のバース·アプリケーションにおける平滑筋機能の測定

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

イメージングローカルCA<sup&gt; 2+</sup&gt;培養哺乳動物細胞内シグナル

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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 ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

FRETベースの共焦点イメージングを使用した培養平滑筋細胞の小胞体内カルシウム変動の測定

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

マウス冠動脈血管平滑筋細胞の単離

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

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 ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

ネズミ大動脈挫傷：効率的<em&gt;インビボ</em&gt;平滑筋細胞の増殖と内皮機​​能のモデル

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...