この作品は、国立衛生研究所からの助成金（JC〜P30のHL101302とRO1 HL109439）とアメリカ心臓協会（JCへSDG 5270005）によって部分的にサポートされていました。 A. BaraziaはT32HL007829 NIHの訓練助成金によって支えられている。

1。生体内顕微鏡の調製（図1A） <ol><li>灌流バッファー（125 mMのNaCl、4.5 mMのKClを、2.5 mMのCaCl 2、1mMのMgCl 2、17 mMの飽和NaHCO 3、pH7.4）を準備します。 </li><li> 37℃バッファおよび熱制御毛布の温度を維持するために、循環水浴℃にオンにします窒素ガス（5％CO 2が窒素とのバランス）でバッファを通気する。 </li><li>顕微鏡システム（サッターラムダDG-4高速波長チェンジャー、ワークステーションコンピュータ、オリンパスBX61W顕微鏡は、MPC-200マルチマニピュレータ、ROE-200ステージコントローラ、高速カメラ、インテンシファイア）をオンにします。 </li><li>血管炎症モデルでは、100パーセントダイクロイックミラーを使用しています。レーザー損傷による血栓形成を誘導するために、50/50％のミラーと100％のミラーを交換し、micropointレーザーアブレーションシステムの電源をオンにします。 </li></ol> 2。生体顕微鏡用の精巣挙筋の調製（図1B） <ol><li>麻酔をかけるケタミン（125 mg / kg体重（BW））およびキシラジン（12.5 mg / kg体重）の腹腔内注射により雄マウス（6-8週齢のC57BL / 6）。イリノイ州インスティテュー動物実験委員会の大学はすべての動物のケアと実験手順を承認した。血管炎症モデルでは、（手術前に2から2.5時間）前の画像へintrascrotallyマウス3時間にマウスTNF-α（250μlの生理食塩水で0.5μg）を注入します。 </li><li>生体顕微鏡トレイに37℃の熱制御の毛布の上にマウスを置きます。 </li><li>鉗子を使用して首の表面に皮を上に引いて、ハサミで1cmに水平に首の皮膚の正中線を切開。 </li><li>優しく鈍はさみを使用して子宮頸部の筋肉を開き、気管を取り巻く筋肉を削除します。 </li><li>呼吸困難を排除するための気管内チューブPE90カニューレを挿入。 </li><li>優しく頸部筋肉の左側を削除し、周囲の組織から頚静脈を隔離する。 </li><li> Cアンヌ抗体および追加の麻酔薬の注入のための左頸静脈に遅くPE10チューブ。 </li><li>優しく引き出し、水平に陰嚢の皮膚を切開する。実験を通して組織の完全性を維持するためには、あらかじめ温めておいたバッファは手術野の上に灌流した。 </li><li> 1鉗子と下腹部を押して、慎重に他の鉗子と睾丸を引き出します。 </li><li>精巣の周りに周囲の結合組織を削除します。 </li><li>垂直精巣挙筋を切開し、周囲を固定することによって、生体内顕微鏡トレイ上のカバーガラスの上に筋肉を平らにする。 </li><li>筋肉のために10〜20分は、データ収集の前に安定させることができます。 30から45μmの直径を有する精巣挙筋細動脈（血栓形成）と細静脈は（血管の炎症のために）我々の研究のために選択された。 </li><li>麻酔状態を維持するために、頸静cannulusを通して約30分毎に同じ麻酔薬の30-50 mlを吹き込む。 </li></ol> &lt;Pクラス= &quot;jove_title&quot;&gt; 3。 TNF-α誘導血管炎症のために生体顕微鏡<ol><li>生体顕微鏡、オープンSlideBook 5.0ソフトウェア上でマウスを配置した後にして、適切な光学系と対物レンズ（60X）を設定するには、メニューバー上の &quot;フォーカス·ウィンドウ&quot;をクリックしてください。 </li><li> Dylightを吹き込む488結合ラット抗マウスCD42c（100μlの生理食塩水で0.1μg/ gでBW）とAlexa Fluor 647-結合ラット抗マウスGR-1抗体（100μlの生理食塩水に0.05μg/ gでBW）の頸静脈を介しcannulus 。 </li><li>メニューバーに移動し、ツールバーの &quot;イメージキャプチャ&quot;をクリックしてください。 </li><li> &quot;イメージ&quot;は、1x1のように &quot;ビン·ファクター&quot;を選択し、高さの幅と &quot;460&quot;の &quot;640&quot;を選択し、新しいメニューがポップアップ表示されます。 </li><li> &quot;フィルタ設定&quot;で、それぞれの露光時間を公開して、設定したいチャンネルのチェックボックスをマークします。たとえば、オープンチャネル：10ミリ、FITCチャンネル：50ミリ秒、およびCy5チャンネル：50ミリ。 </li><li>時点と間隔の数を調整する &quot;タイムラプス撮影&quot;を選択します。フォーR炎症モデル、時間ポイントの数は、200ミリ秒（5分合計経過時間）の間隔で1,000〜1,500に設定されています。 </li><li>そのチャネルに指定された露光時間でシングルプレーンのイメージを表示するには &quot;Test&quot;をクリックします。ヒストグラムが適切な強度分布を（マルチチャンネル·キャプチャでは、各チャンネルごとに、このプロセスを繰り返します）が表示されるまでの露光時間および/または顕微鏡光を調整します。 </li><li>一度すべてのパラメータは、 &quot;イメージキャプチャ&quot;で（チャンネル、露光時間など）が設定されて、キャプチャを開始するには &quot;スタート&quot;を選択します。 </li><li>血管炎症モデルにおける炎症精巣挙小静脈の上半分に5分間圧延および付着血小板や好中球を監視します。八から十異なる細静脈は、1つのマウスに記録されます。 </li><li>画像は157×118μmのμmのウィンドウサイズを与える60Xの総合倍率（1.0 NAの水浸対物レンズ）を使用して撮影した。 </li><li>実験終了時には、マウスを頸椎dislocatio経由で安楽死させたnである。 </li></ol> 4。細静脈の炎症モデルのためのデータ解析<ol><li> SlideBook 5.0ソフトウェアを開き、ファイルを開くには、メニューバーの &quot;フォルダ&quot;をクリックしてください。 </li><li>ドロップダウンチャンネルメニューをクリックし、 &quot;オープン、FITC、Cy5で、等&quot;を選択してください</li><li> &quot;（赤と緑のバー）再正規化&quot;をクリックしてチャンネルを選択します。大体蛍光シグナルを最適化するために、左または右に赤と緑のバーをドラッグします。 </li><li> &quot;適用&quot;と &quot;OK&quot;画像を更新します。 </li><li>デフォルトとして、現在の表示を選択する &quot;サムネイルボタン&quot;をクリックします。 </li><li>キャプチャされたタイムラプス画像を再生し、5分間の間圧延および接着性好中球を数える。 </li><li> 血小板血栓形成を分析するには 、 &quot;マスク&quot;に行って、 &quot;作成&quot;を選択します。 </li><li> &quot;現在のイメージでは&quot;マスクとマークを付けます。 </li><li>メインビューで &quot;矩形選択ツール&quot;の &quot;大規模な鉛筆アイコン&quot;をクリックしてください。 </li><li>背景マスクを設定するには、メインビューに容器の外側の領域に色をつける。 </li><li> tを行く○ &quot;マスク&quot;、 &quot;この飛行機をコピー&quot;をクリックし、 &quot;現在の時点でコピーマスク&quot;をクリックし、 &quot;すべての時点&quot;を選択し、 &quot;OK&quot;をクリックします。 </li><li> 背景信号を計算するには 、 &quot;統計&quot;に移動し、 &quot;マスクの統計&quot;をクリックします。 </li><li>イメージスコープで &quot;現在の2D時間の経過または4D画像（s）&quot;をクリックして、マスクの範囲内の &quot;マスク全体&quot;を選択します。特集では、 &quot;経過時間（HH：MM：ミリ秒）&quot;を選択してキャプチャの日付の下に、選択した &quot;エリア（ピクセル）&quot;形態計測で、強度の下で &quot;最大輝度&quot;を選択し、 &quot;エクスポート&quot;をクリック（この統計方法は、私たちにでき自家蛍光強度（バックグラウンド信号））を計算します。 </li><li> MS OfficeのExcelでテキストフ​​ァイルを開き、記録期間を通じてバックグラウンド信号の平均値を計算します。これは、バックグラウンド蛍光強度である。 </li><li> 血栓強度を決定するには 、メインビューで&quot;矩形選択ツール&quot;で再び&quot;大鉛筆アイコン&quot;をクリックしてください。 </li><li>血小板信号を設定するためのメインビューで、容器内の色。 </li><li> &quot;マスク&quot;に進み、 &quot;この飛行機をコピー&quot;をクリックし、 &quot;現在の時点でのコピーマスク&quot;をクリックし、 &quot;すべての時点&quot;を選択し、 &quot;OK&quot;をクリックします。 </li><li> &quot;統計&quot;に移動し、 &quot;マスクの統計&quot;をクリックします。 </li><li>イメージスコープで &quot;現在の2D時間の経過または4D画像（s）&quot;をクリックして、マスクの範囲内の &quot;マスク全体&quot;を選択します。機能では、選択して &quot;経過時間（HH：MM：MS）の&quot;キャプチャーの日付の下に、私たちにできるという &quot;面積（画素）&quot;形態計測で、強度の下で &quot;合計強度&quot;を選択し、 &quot;エクスポート&quot;（この統計方法をクリックし選択）容器内の蛍光強度を計算します。 </li><li> MS OfficeのExcelでテキストフ​​ァイルを開きます。血小板の蛍光シグナルは、血管の内側の和の強度からバックグラウンド強度×面積（画素）の平均値を減算することによって計算されます。これは血小板血栓の蛍光強度である。 </li><li> 3〜5つの異なったマウスでは30種類の細静脈でこれを繰り返します。その後、血小板の蛍光強度の中央値を計算する。 </li><li> へ好中球のローリングとの接着性を分析し、好中球の転がり流入は各小静脈で5分間にわたって決定し、毎分の細胞数として発表された。 &gt; 30秒間静止していたゆっくりとクロール（&lt;10μmの30秒以上）がロールオーバーされませんでした付着性好中球数は、5分ごとの細胞数としてカウントされ、発表されました。 </li></ol> 5。レーザー誘起動脈血栓症に対する生体顕微鏡アブレーションレーザー5-1キャリブレーション<ol><li>顕微鏡ステージ上でミラーリングされたスライドを入れ、スライドの上部に蒸留水の小滴を適用し、強度を調整して、接眼レンズを使用して焦点を当てています。 </li><li> &quot;フォーカスウィンドウ&quot;を開き、SlideBook 5.0ソフトウェアでは &quot;FRAP&quot;タブを選択します。 </li><li> 40から50にレーザパワーを設定し、両方の &quot;ダブルクリックの繰り返し&quot;と &quot;ダブルクリックサイズ&quot; 8に設定します。クロスヘアのセットを起動するには、 &quot;ガイド&quot;ボックスをマークします。 </li><li>タスクバーoから &quot;矢印&quot;アイコンを選択nは画面の上部に。 </li><li> &quot;FRAPアライメント&quot;タブで、 &quot;次の火&quot;をクリックしてください。小さなスポットは、モニターの左下隅の近くに表示されるはずです。スポットが表示されたら、それの中心をダブルクリックします。一度クリックすると、別の場所は、上記に表示され、最初のスポットの右側にする必要があり、それをダブルクリックします。キャリブレーションパラメータを更新するために完了したら、 &quot;Save&quot;をクリックします（16 番目のスポットは、右下隅に表示されます。）16点に対してこの手順を繰り返します。 </li><li>キャリブレーションの精度をテストするには、画面の中央に表示されるはずです &quot;センター&quot;ボタン小さなスポットをクリックしてください。されていない場合は、所望の精度が達成されるまで、校正を繰り返す。 </li></ol> 5月2日レーザー誘起細動脈の血栓形成<ol><li>顕微鏡ステージ上でマウスを麻酔し（2-2〜2-12の手順を参照してください）​​を配置します。 </li><li> &quot;キャプチャ&quot;ウィンドウの下のプロトコルを選択するか、または新しいものを作成します。設定は次のとおりです。CY5（70ミリ秒露光）、オープン（10 MSEC露出）、およびFITC（50ミリ秒露光）。画像は20分（500ミリ秒のインターバル時間が約2400時間点）上に捕捉される。 </li><li> Dylight前述のように488結合抗マウスCD42cおよびAlexa Fluor 647標識抗マウスGR-1抗体を吹き込む。 </li><li> 3-7を3-3で説明されているように、蛍光強度と、明視野顕微鏡像を含むパラメータを最適化します。 </li><li>適切な明視野強度や抗体シグナル、動脈配置し、フォーカスを確保するための &quot;テスト&quot;ボタンをクリックします。 </li><li>イメージキャプチャプロセスを開始するために &quot;Start&quot;をクリックしてください。 </li><li>アブレーションレーザーを発射する前に、マウスポインターのアイコンが選択され、血管壁に明確な焦点になっていることをされていることを確認してください。 </li><li>レーザーを発射するには、二重容器壁2から3μmの内部でスポットをクリックしてください。細動脈内皮細胞の傷害が明視野像では明らかなはずである視覚的形状の変更が生じますが、血小板の蓄積が続く。モニター血栓レーザー損傷後の5分間形成。 </li><li>レーザー損傷5分後に一時停止（血栓サイズは、現在安定していなければならない）とキャプチャをキャンセルします。その後、20分間付着血小板やローリング/付着した好中球を記録するために500ミリ秒間隔で2400年時点でキャプチャを開始。 3〜5個の動脈は、1つのマウスに記録されます。 </li><li>実験終了時には、マウスを頸椎脱臼を経由して安楽死させた。 </li></ol> 6。動脈血栓モデルのためのデータ解析<ol><li> 4.5〜ステップ4.1を越える。 </li><li> 血小板血栓形成中にバックグラウンド蛍光強度を得るためには 、4.14〜ステップ4.7を越える。 </li><li> &quot;マスク&quot;に進み、 &quot;セグメント&quot;をクリックして、チャネル内のFITCを選択して、ローに背景信号の平均値を挿入します。 &quot;適用&quot;をクリックし、 &quot;OK&quot;をクリックします。 </li><li> 血小板血栓の蛍光強度（Dylight 488結合抗CD42c抗体）を分析するために、 </stチョロン&gt; 4.19〜ステップ4.18を越える。抗体シグナルを計算するには、 &quot;合計強度が&quot;バックグラウンド信号（ &quot;最大輝度&quot; x &quot;の面積（ピクセル）&quot;）から減算されます。これは血小板血栓の蛍光強度である。 </li><li> 圧延および好中球の接着を定量化するために 、微速度撮影を再生し、目に見えて20分かけて血小板血栓をロールオーバーしたセルの数をカウントします。少なくとも2秒間血小板血栓と対話しながらのローリングは、好中球の速度の低下として定義されています。付着した好中球は、少なくとも2分間血小板血栓に接続されたまま、任意の好中球のように定義されます。ローリングと接着好中球数は、20分あたりの細胞数として発表された。ローリング速度は、粒子追跡システムを用いて算出した。 </li></ol>

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C., Merrill-Skoloff, G., Chou, J., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-versus+microparticle-mediated+tissue+factor+transfer+during+arteriolar+thrombus+development.">Leukocyte-versus microparticle-mediated tissue factor transfer during arteriolar thrombus development.</a> Journal of Leukocyte Biology. 78, 1318-1326 (2005).</li><li>Falati, S., Gross, P., Merrill-Skoloff, G., Furie, B. C., Furie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+in+vivo+imaging+of+platelets,+tissue+factor+and+fibrin+during+arterial+thrombus+formation+in+the+mouse.">Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse.</a> Nat. Med. 8, 1175-1181 (2002).</li><li>Barthel, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha+1,3+fucosyltransferases+are+master+regulators+of+prostate+cancer+cell+trafficking.">Alpha 1,3 fucosyltransferases are master regulators of prostate cancer cell trafficking.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 19491-19496 (2009).</li><li>Trzpis, M., McLaughlin, P. M., de Leij, L. M., Harmsen, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+adhesion+molecule:+more+than+a+carcinoma+marker+and+adhesion+molecule.">Epithelial cell adhesion molecule: more than a carcinoma marker and adhesion molecule.</a> The American Journal of Pathology. 171, 386-395 (2007).</li><li>Junt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+visualization+of+thrombopoiesis+within+bone+marrow.">Dynamic visualization of thrombopoiesis within bone marrow.</a> Science. 317, 1767-1770 (2007).</li><li>Egan, C. E., Sukhumavasi, W., Bierly, A. L., Denkers, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+multiple+functions+of+Gr-1(+)+cell+subpopulations+during+microbial+infection.">Understanding the multiple functions of Gr-1(+) cell subpopulations during microbial infection.</a> Immunologic Research. 40, 35-48 (2008).</li></ol>

著者らは競合する経済的利害関係を宣言しない。

ここでは、血管の炎症や血栓症の間に活性化内皮上の異血小板、好中球の相互作用を可視化するためのリアルタイム蛍光生体顕微鏡検査のための詳細なプロトコルを記述します。以前、似たような蛍光顕微鏡のアプローチは血栓形成と血管炎症の分子メカニズムを研究することが報告された。8,12異型細胞間の相互作用は、損傷部位での血管閉塞のために重要であるかもしれないので?...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>7447-40-7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2 2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>10035-04-8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2 6H2O</td>
			<td>Fisher Scientific</td>
			<td>7791-18-6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher Scientific</td>
			<td>144-55-8</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.9% NaCl Saline</td>
			<td>Hospira</td>
			<td>0409-4888-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Hospira</td>
			<td>0409-2051-05</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Lloyd</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 90)</td>
			<td>BD Diagnostics</td>
			<td>427421</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intramedic Tubing (PE 10)</td>
			<td>BD Diagnostics</td>
			<td>427401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Murine TNF-&alpha;</td>
			<td>R&amp;D Systems</td>
			<td>410-MT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dylight 488- labeled rat anti-mouse CD42b antibody</td>
			<td>Emfret Analytics</td>
			<td>X488</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody</td>
			<td>BioLegend</td>
			<td>108418</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NESLAB EX water bath/circulator</td>
			<td>Thermo-Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olympus BX61W microscope</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TH4-100 Power</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lambda DG-4</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MPC-200 multi-manipulator</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ROE-200 stage controller</td>
			<td>Sutter</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>C9300 high-speed camera</td>
			<td>Hamamatsu</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Intensifier</td>
			<td>Video Scope International</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ablation Laser</td>
			<td>Photonic Instruments, Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SlideBook 5.0</td>
			<td>Intelligent Imaging Innovations</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

real-time imaging of heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice

活性化された内皮細胞を媒介血栓症および血管炎症で活性化された血小板と白血球の相互作用（主に好中球）。1,2動脈損傷部位に血栓形成の間、活性化内皮に付着した血小板と内皮下マトリックスタンパク質は、好中球のローリングや接着をサポートしています3逆に、細静脈の炎症性条件の下で、活性化された内皮細胞に付着して好中球が循環する血小板の付着·蓄積をサポートすることができます。異血小板好中球凝集は細胞間の特異的受容体-カウンター受容体の相互作用によってシーケンシャルプロセスを必要とします。4それが活性化された内皮細胞は、それによって高剪断条件下で血小板の接着および蓄積を開始する、そのようなvon Willebrand因子として接着分子を放出することが知られている。5はまた、活性化された内皮細胞は、セレクチン発現させることにより、好中球のローリングとの接着性をサポートD細胞間接着分子-1（ICAM-1）は、それぞれ、低剪断条件下で4血小板P-セレクチンは通って好中球と相互作用するP-セレクチン糖タンパク質リガンド-1（PSGL-1）することにより、好中球β2インテグリンと企業の活性化を誘導する2種類の細胞間の接着。異型の血小板、好中球の相互作用は、全血または単離された細胞、6,7で決定されたin vitroの実験での進歩にもかかわらず、それらの研究では、血管疾患の間に酸化ストレス状態を操作することはできません。本報告では、蛍光標識を使用して、マウス血小板および好中球のマーカーに対する特異的抗体は、我々はTNF-α誘導炎症時に活性化された内皮細胞に血小板や好中球の異型相互作用をモニターするための詳細な生体顕微鏡プロトコルを記述したり、レーザー誘起に続く生きたマウスの精巣挙筋の微小血管の損傷。

詳細な生体顕微鏡分析を用いて、活性化された内皮細胞上の異型の血小板、好中球の相互作用は、生きたマウスへ血小板（CD42c）または好中球マーカー（GR-1）に対する蛍光標識抗体の注入による可視化した。 TNF-α誘導細静脈の炎症のモデルでは、最も転がり好中球は安定記録期間（TNF-αの注射後3から4.5時間、 図2Aの間に、ICAM-1の活性化β2インテグリンとの?...

Watch this Scientific Journal Video about Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mic

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

ここでは、血管の炎症や生きたマウスにおける血栓形成の間に活性化内皮上の異血小板、好中球の相互作用を可視化する蛍光生体顕微鏡の実験手法を報告する。この顕微鏡技術は、血管疾患の分子メカニズムを研究することが、病態生理学的な条件下での薬理学的薬剤をテストするためにも役立ちます。

生きたマウスにおける血管炎症及び血栓形成の間に活性化内皮上に異型血小板、好中球の相互作用のリアルタイムイメージング

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

real-time-imaging-heterotypic-platelet-neutrophil-interactions-on

Research

JoVE Journal

Immunology and Infection

15.6K ビュー.  University of Illinois at Chicago. ここでは、血管の炎症や生きたマウスにおける血栓形成の間に活性化内皮上の異血小板、好中球の相互作用を可視化する蛍光生体顕微鏡の実験手法を報告する。この顕微鏡技術は、血管疾患の分子メカニズムを研究することが、病態生理学的な条件下での薬理学的薬剤をテストするためにも役立ちます。

ビデオ: Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

ロイコトリエンBのリアルタイムイメージング 4介在細胞の移行とβ-アレスチンとBLT1の相互作用

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

免疫・感染学

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

高腔内圧で血管内皮へのイメージングの白血球接着

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

ノーマルCNSのリアルタイムPCRによると神経炎症時のミクログリアにおけるマイクロRNAの検出

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

複合体形成シグナリングT細胞のリアルタイムのライブイメージング

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

好中球遊出の下で生理的流れの間に内皮細胞 - 細胞接合のリアルタイムイメージング

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

小説<em&gt;インビトロ</em&gt;ヒト全血と内皮の間の相互作用を研究するためのモデル

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

イメージング病因とのコーディングを解読し、<em&gt;マイコバクテリウム膿瘍</em&gt;ゼブラフィッシュ胚で

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

リアルタイムで麻酔したマウスに生体顕微鏡により腸間膜静脈に好中球および単球のイメージング

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

リアルタイム細胞外フラックス分析を用いた偏光M1、M2の骨髄由来マクロファージの代謝特性評価

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

全血-内皮相互作用と血栓動態をリアルタイムで研究するインビトロ微小流体疾患モデル

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...