この作品は、麻酔と周術期ケアのUCSF学科によってサポートされていました。

1。微小血管内皮細胞のメッキとメンテナンス<ol><li>命令を供給メーカーによるとあなたの微小血管内皮細胞を解凍し、成長する。このプロトコルでは、細胞をEGM-2 MVメディア（ロンザ）で増殖させた、そしてそれは全ての実験は、継代数に起因するいかなる変動を最小限に抑えるために解凍から2週間以内に行われることが推奨される。 HMVECロンと私たちの実験は4〜9の通路との間で行われている。 </li><li> 1日目：細胞が80〜90％コンフルエントに達したとき、トリプシン処理及び1ml当たり120,000細胞で再懸濁します。 48ウェル、ポリスチレン組織培養プレート（S）にプレートウェルあたり36,000細胞（0.3ミリリットル）を。バックグラウンド蛍光測定値を得るために、好中球とインキュベートされることはありません HMVECの井戸を含めます。特に蛍光アッセイ、交互の行または列用に設計された48ウェルプレートを用いた場合を除き、隣接する井戸からの光の汚染を最小限に抑えることができます。注：ウェルズポリlysiでプレコートすることができますね、コラーゲンやフィブロネクチン。 </li><li> 2日目：新鮮な培地を追加します。 </li><li> 3日目：位相差顕微鏡で、細胞（ すなわち 、 &quot;玉石&quot;の外観と少し細胞の破片）健康的であり、彼らが100％コンフルエント（ 図1）であることを確認します。細胞が100％コンフルエントでない場合、彼らは準備が整うまで、毎日メディアを変更してください。 </li><li> HMVEC単層が治療のために準備ができたら、ハンクス平衡塩溶液（HBSS）で1回細胞を洗浄し、適切なウェルに炎症性アゴニストを含有する新鮮なEGM-2 MVメディアまたはメディア0.3 mlを加える。このプロトコルではHMVEC-肺をTNFαで処理した3時間[100 ngの/ mlの]（ぺプロテック、ニュージャージー州）、これはECの21のよく説明ベーターであるため。注：これは、（ステップ3.5）、好中球のラベルが付いたAM、時間あなたの実験をので、あなたの活性化HMVEC細胞（ステップ4.1）とカルセインことをことをお勧めしますが、同時に使用する準備が整いました。それはneutrophを分離し、標識するために私たちに約2.5時間かかりますILS。 </li></ol> 2。 Polymorphprepを使用して好中球分離<ol><li>以前に22説明したように好中球を分離し、または全血から多形核顆粒球を分離するため、既製の密度勾配液で。 Nuzzi、 らによるプロトコルに従ってPolymorphprepを採用。全血23 1mlあたり約2〜3×10 6の好中球の利回り2007年の結果は。注：赤血球を除去するために過剰な赤血球溶解、デキストラン沈降を回避するためには、密度勾配遠心24の前に行ってもよい。 </li><li> RTへPolymorphprep密度勾配液を持参してください。 </li><li>例えば、ヘパリン、クエン酸塩またはEDTAなどの抗凝固剤の存在下で、健康な人間のボランティアからの全血30mlのを収集します。血液は、2時間以内に使用され、18-22℃の間で保管してください</li><li> 15ミリリットルコニカルチューブ内の密度勾配溶液5mlを超える全血の層5ミリリットル（ <strong&gt;図2A）。これは、その後の遠心分離条件を変更することができるように血液および密度勾配溶液の容積を変化させる避ける。 </li><li> 18-22で30分間450×gで遠心℃、ゆっくりスピードアップとダウンの遠心分離機（ すなわち遠心ブレーキをオフにする）、および好中球の活性化を減少させ、層の混合を防ぐために振動を防ぐためによくチューブのバランスをとることを確認してください。長い遠心倍以上の遠心力がペレット化赤血球に向かってさらに下方に移動するように、好中球を含む下位の白血球層をもたらす。 </li><li>好中球（ 図2B）を含む層の汚染を防止するために、PBMCを含有プラズマ及び上部白血球バンドを吸引。 </li><li>好中球を含む白血球下位層を除去するために1〜2ミリリットル血清ピペットを使用してください。 50mlのPBSを30ml（のCa 2 +およびMg 2 +なし）に好中球のレイヤーを結合コニカルチューブ。 </li><li> PBSで50ミリリットル（のCa 2 +およびMg 2 +なし）にボリュームを起動し、18〜22歳で10分間、450×gで遠心℃、 </li></ol> 注：ステップ2.9と2.10はオプションですが、強く推奨。 <ol start="9"><li>上清を吸引。赤血球を溶解するために30秒間の滅菌水8mlに好中球を再懸濁し、すぐに5倍の2mlのPBS（のCa 2なし+およびMg 2 +）と優しく手でミックス。で細 ​​胞をインキュベートしない]に細胞懸濁液を追加30秒より長くのための水の重要な好中球の死が発生する可能性があるため。 </li><li> 18-22で5分間250 xgで遠心℃、 </li><li> 10mlのPBS（Ca 2 +のおよびMg 2 +なし）で1回細胞を洗浄し、18〜22歳で5分間250 xgで再び遠心℃、 </li><li> RPMI-1640（フェノールレッドなし）10ml中の好中球を再懸濁し、トリパンブルーを用いて生細胞を数える電子色素排除法。これは、好中球の活性化につながる可能性があるので、1mlあたり5×10 6より大きい細胞密度の長時間は避けてください。 </li></ol> 3。カルセインAMと好中球のラベル<ol><li> 6×10 5好中球は、48ウェルプレートのウェルあたりに使用される。 </li><li>カウントした後、50ミリリットルコニカルチューブに再懸濁した細胞を転送し、フェノールレッドを含まないRPMI-1640で1mlあたり2-4×10 6細胞に好中球を希釈。 </li><li>カルセインが3μm（カルセインAM株式すなわち 2.98液（1 mg / ml）でml当たりのメディア）に原液AM加え、穏やかにチューブをフリックでよく混ぜる。以上5μMはカルセイン好中球からの漏れが発生しますAM 注：非蛍光カルセインAMが高い蛍光分子カルセイン（ つまり死んだ細胞が蛍光を発することはありません）を生成するために、内因性エステラーゼにより細胞内で切断される。 </li><li>アルミホイルでチューブをカバーとfをインキュベートまたは37℃で30分間、暗注C：長いインキュベーション時間は接着アッセイの過程で好中球から解放され、よりカルセインになることがあります。 </li><li> 18-22°Cで5分間450×gで遠心分離し、RPMI-1640（フェノールレッドなし、37℃に予熱）10mlで2倍の好中球を洗う。 40μM無菌フィルターは18-22で塊を除去した後、5分間450 xgで再び遠心分離するのに二回目の洗浄のために細胞を再懸濁した後、最初の好中球を渡​​す℃に</li><li> 1mlあたり2×10 6好中球で。（37℃に予め温めずにフェノールレッド）RPMI 1640中で好中球を再懸濁し</li></ol> 4。好中球結合アッセイ<ol><li>フィルター滅菌を0.5ml（0.22μm）をRPMI 1640（フェノールレッドなし）ウェルあたり3％BSAを含むと3倍HMVEC単層を洗浄します。 </li><li>メディアを吸引し、6×10 5カルセイン標識好中球（細胞サスペンシオ0.3 mlを加える48ウェルプレートの適当なウェルに好中球なしN）、またはRPMI 1640 0.3mlの（フェノールレッドなし）、 注：これは1cm 2あたり8×10 5の好中球と同等です。 </li><li> 37°C、5％CO 2インキュベーター内で20分間インキュベートする。 </li><li> 総好中球蛍光（PRE洗浄）：485 nmでの励起波長および発光FLUOstarにおける520nmの（フルオレセインフィルターセット）の波長、またはそれと同等の分光光度計を用いて各ウェルの蛍光強度を測定する。直前のインキュベーション終了点までこのステップを実行注：このプロトコルでは、放出される光のカルセインの励起および検出が覆わウェルの上部から行われたが、両方はまた、ウェルの底部から行うことができる。 </li><li> 0.5ミリリットルウェルあたりのPBS（W / Ca 2 +のおよびMg 2 +）と5倍HMVEC単層および好中球を含むウェルを洗ってください。メディアや洗浄を削除反転プレートによって、残りの液体を除去するためにペーパータオルの上に優しくパッティング。 </li><li>バックウェルあたりRPMI 1640 0.3ミリリットル（フェノールレッドなし）を追加します。 </li><li> 付着性好中球蛍光（POSTウォッシュ）：ステップ4.4のように、各ウェルの蛍光強度を測定します。 </li><li>パーセント順守とウェルあたり相対遵守を決定する： </li></ol> 注：この段階で、カルセイン-標識された好中球の画像は標準フルオレセインフィルターを備えた蛍光顕微鏡を使用することができることによって得ることができる。 図4の画像は、Pro7ソフトウェア（Qイメージング）、Qは-キャプチャ使用Retiga 2000Rカメラ（Qイメージング）を搭載したオリンパスIX51倒立顕微鏡で得た。

<ol>
	<li>Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+the+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: the leukocyte adhesion cascade updated.</a> Nat. Rev. Immunol. 7, 678-689 (2007).</li><li>Collins, 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=Transcriptional+regulation+of+endothelial+cell+adhesion+molecules:+NF-kappa+B+and+cytokine-inducible+enhancers.">Transcriptional regulation of endothelial cell adhesion molecules: NF-kappa B and cytokine-inducible enhancers.</a> FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. 9, 899-909 (1995).</li><li>Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+physiological+roles+of+inflammation.">Origin and physiological roles of inflammation.</a> Nature. 454, 428-435 (2008).</li><li>Chen, G. Y., Nunez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterile+inflammation:+sensing+and+reacting+to+damage.">Sterile inflammation: sensing and reacting to damage.</a> Nat. Rev. Immunol. 10, 826-837 (2010).</li><li>Middleton, J., 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=Transcytosis+and+surface+presentation+of+IL-8+by+venular+endothelial+cells.">Transcytosis and surface presentation of IL-8 by venular endothelial cells.</a> Cell. 91, 385-395 (1997).</li><li>Rot, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+binding+of+NAP-1/IL-8:+role+in+neutrophil+emigration.">Endothelial cell binding of NAP-1/IL-8: role in neutrophil emigration.</a> Immunol. Today. 13, 291-294 (1992).</li><li>Detmers, P. A., 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=Neutrophil-activating+protein+1/interleukin+8+stimulates+the+binding+activity+of+the+leukocyte+adhesion+receptor+CD11b/CD18+on+human+neutrophils.">Neutrophil-activating protein 1/interleukin 8 stimulates the binding activity of the leukocyte adhesion receptor CD11b/CD18 on human neutrophils.</a> J. Exp. Med. 171, 1155-1162 (1990).</li><li>Laudanna, C., Kim, J. Y., Constantin, G., Butcher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+leukocyte+integrin+activation+by+chemokines.">Rapid leukocyte integrin activation by chemokines.</a> Immunol. Rev. 186, 37-46 (2002).</li><li>Simon, S. I., Hu, Y., Vestweber, D., Smith, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+tethering+on+E-selectin+activates+beta+2+integrin+binding+to+ICAM-1+through+a+mitogen-activated+protein+kinase+signal+transduction+pathway.">Neutrophil tethering on E-selectin activates beta 2 integrin binding to ICAM-1 through a mitogen-activated protein kinase signal transduction pathway.</a> J. Immunol. 164, 4348-4358 (2000).</li><li>Zarbock, A., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+consequences+of+neutrophil+interaction+with+the+endothelium.">Mechanisms and consequences of neutrophil interaction with the endothelium.</a> The American Journal of Pathology. 172, 1-7 (2008).</li><li>Muller, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+leukocyte+transendothelial+migration.">Mechanisms of leukocyte transendothelial migration.</a> Annu. Rev. Pathol. 6, 323-344 (2011).</li><li>Brinkmann, V., 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=Neutrophil+extracellular+traps+kill+bacteria.">Neutrophil extracellular traps kill bacteria.</a> Science. 303, 1532-1535 (2004).</li><li>Kumar, V., Sharma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils:+Cinderella+of+innate+immune+system.">Neutrophils: Cinderella of innate immune system.</a> International Immunopharmacology. 10, 1325-1334 (2010).</li><li>McDonald, B., 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=Intravascular+danger+signals+guide+neutrophils+to+sites+of+sterile+inflammation.">Intravascular danger signals guide neutrophils to sites of sterile inflammation.</a> Science. 330, 362-366 (2010).</li><li>Nathan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+and+immunity:+challenges+and+opportunities.">Neutrophils and immunity: challenges and opportunities.</a> Nat. Rev. Immunol. 6, 173-182 (2006).</li><li>Soehnlein, O., Lindbom, L., Weber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+underlying+neutrophil-mediated+monocyte+recruitment.">Mechanisms underlying neutrophil-mediated monocyte recruitment.</a> Blood. 114, 4613-4623 (2009).</li><li>Wilhelmsen, K., Mesa, K. R., Prakash, A., Xu, F., Hellman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+endothelial+TLR2+by+bacterial+lipoprotein+upregulates+proteins+specific+for+the+neutrophil+response.">Activation of endothelial TLR2 by bacterial lipoprotein upregulates proteins specific for the neutrophil response.</a> Innate Immun. 18, 602-616 (2012).</li><li>Haugland, R. P., Johnson, I. D., Basey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Handbook: A Guide to Fluorescent Probes and Labelling Technologies. , (2005).</li><li>Shin, H. S., 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=Bacterial+lipoprotein+TLR2+agonists+broadly+modulate+endothelial+function+and+coagulation+pathways+in+vitro+and+in+vivo.">Bacterial lipoprotein TLR2 agonists broadly modulate endothelial function and coagulation pathways in vitro and in vivo.</a> J. Immunol. 186, 1119-1130 (2011).</li><li>Wilhelmsen, K., Mesa, K. R., Lucero, J., Xu, F., Hellman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERK5+protein+promotes,+whereas+MEK1+protein+differentially+regulates,+the+Toll-like+receptor+2+protein-dependent+activation+of+human+endothelial+cells+and+monocytes.">ERK5 protein promotes, whereas MEK1 protein differentially regulates, the Toll-like receptor 2 protein-dependent activation of human endothelial cells and monocytes.</a> J. Biol. Chem. 287, 26478-26494 (2012).</li><li>Pober, J. S., Sessa, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolving+functions+of+endothelial+cells+in+inflammation.">Evolving functions of endothelial cells in inflammation.</a> Nat. Rev. Immunol. 7, 803-815 (2007).</li><li>Oh, H., Siano, B., Diamond, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+isolation+protocol.">Neutrophil isolation protocol.</a> J. Vis. Exp. (17), e745 (2008).</li><li>Nuzzi, P. A., Lokuta, M. A., Huttenlocher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+neutrophil+chemotaxis.">Analysis of neutrophil chemotaxis.</a> Methods Mol. Biol. 370, 23-36 (2007).</li><li>Garcia-Garcia, E., Uribe-Querol, E., Rosales, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+efficient+method+to+detect+nuclear+factor+activation+in+human+neutrophils+by+flow+cytometry.">A simple and efficient method to detect nuclear factor activation in human neutrophils by flow cytometry.</a> J. Vis. Exp. (74), e50410 (2013).</li><li>Kuma, Y., 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=BIRB796+inhibits+all+p38+MAPK+isoforms+in+vitro+and+in+vivo.">BIRB796 inhibits all p38 MAPK isoforms in vitro and in vivo.</a> J. Biol. Chem. 280, 19472-19479 (2005).</li><li>Westra, J., Kuldo, J. M., van Rijswijk, M. H., Molema, G., Limburg, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine+production+and+E-selectin+expression+in+activated+endothelial+cells+are+inhibited+by+p38+MAPK+(mitogen+activated+protein+kinase)+inhibitor+RWJ+67657.">Chemokine production and E-selectin expression in activated endothelial cells are inhibited by p38 MAPK (mitogen activated protein kinase) inhibitor RWJ 67657.</a> International Immunopharmacology. 5, 1259-1269 (2005).</li><li>Leeuwenberg, J. F., Jeunhomme, G. M., Buurman, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+of+polymorphonuclear+cells+to+human+endothelial+cells.+Adhesion-molecule-dependent,+and+Fc+receptor-mediated+adhesion-molecule-independent+mechanisms.">Adhesion of polymorphonuclear cells to human endothelial cells. Adhesion-molecule-dependent, and Fc receptor-mediated adhesion-molecule-independent mechanisms.</a> Clinical and Experimental Immunology. 81, 496-500 (1990).</li><li>Akeson, A. L., Woods, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorometric+assay+for+the+quantitation+of+cell+adherence+to+endothelial+cells.">A fluorometric assay for the quantitation of cell adherence to endothelial cells.</a> Journal of Immunological Methods. 163, 181-185 (1993).</li><li>Vaporciyan, A. A., Jones, M. L., Ward, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+leukocyte-endothelial+adhesion.">Rapid analysis of leukocyte-endothelial adhesion.</a> Journal of Immunological Methods. 159, 93-100 (1993).</li><li>De Clerck, L. S., Bridts, C. H., Mertens, A. M., Moens, M. M., Stevens, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+fluorescent+dyes+in+the+determination+of+adherence+of+human+leucocytes+to+endothelial+cells+and+the+effect+of+fluorochromes+on+cellular+function.">Use of fluorescent dyes in the determination of adherence of human leucocytes to endothelial cells and the effect of fluorochromes on cellular function.</a> Journal of Immunological Methods. 172, 115-124 (1994).</li><li>Bevilacqua, M. P., Pober, J. S., Wheeler, M. E., Cotran, R. S., Gimbrone, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+1+acts+on+cultured+human+vascular+endothelium+to+increase+the+adhesion+of+polymorphonuclear+leukocytes,+monocytes,+and+related+leukocyte+cell+lines.">Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines.</a> The Journal of Clinical Investigation. 76, (1985).</li><li>Schleimer, R. P., Rutledge, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+human+vascular+endothelial+cells+acquire+adhesiveness+for+neutrophils+after+stimulation+with+interleukin+1,+endotoxin,+and+tumor-promoting+phorbol+diesters.">Cultured human vascular endothelial cells acquire adhesiveness for neutrophils after stimulation with interleukin 1, endotoxin, and tumor-promoting phorbol diesters.</a> J. Immunol. 136, 649-654 (1986).</li><li>Gamble, J. R., Harlan, J. M., Klebanoff, S. J., Vadas, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+the+adherence+of+neutrophils+to+umbilical+vein+endothelium+by+human+recombinant+tumor+necrosis+factor.">Stimulation of the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor.</a> Proc. Natl. Acad. Sci. U.S.A. 82, 8667-8671 (1985).</li><li>Braut-Boucher, F., 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=A+non-isotopic,+highly+sensitive,+fluorimetric,+cell-cell+adhesion+microplate+assay+using+calcein+AM-labeled+lymphocytes.">A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes.</a> Journal of Immunological Methods. 178, 41-51 (1995).</li><li>Ait-Oufella, H., Maury, E., Lehoux, S., Guidet, B., Offenstadt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelium:+physiological+functions+and+role+in+microcirculatory+failure+during+severe+sepsis.">The endothelium: physiological functions and role in microcirculatory failure during severe sepsis.</a> Intensive Care Medicine. 36, 1286-1298 (2010).</li><li>Andonegui, G., 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=Endothelium-derived+Toll-like+receptor-4+is+the+key+molecule+in+LPS-induced+neutrophil+sequestration+into+lungs.">Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs.</a> The Journal of Clinical Investigation. 111, 1011-1020 (2003).</li><li>Sharma, J., 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=Lung+endothelial+cell+platelet-activating+factor+production+and+inflammatory+cell+adherence+are+increased+in+response+to+cigarette+smoke+component+exposure.">Lung endothelial cell platelet-activating factor production and inflammatory cell adherence are increased in response to cigarette smoke component exposure.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 302, L47-L55 (2012).</li><li>Deban, L., Correale, C., Vetrano, S., Malesci, A., Danese, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathogenic+roles+of+microvasculature+in+inflammatory+bowel+disease:+a+Jack+of+all+trades.">Multiple pathogenic roles of microvasculature in inflammatory bowel disease: a Jack of all trades.</a> The American Journal of Pathology. 172, 1457-1466 (2008).</li><li>Gross, W. L., Trabandt, A., Csernok, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Wegener&amp;#39;s+granulomatosis.">Pathogenesis of Wegener&amp;#39;s granulomatosis.</a> Annales de Medecine Interne. 149, 280-286 (1998).</li><li>Chen, Y., 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=Evidence+of+inflammatory+cell+involvement+in+brain+arteriovenous+malformations.">Evidence of inflammatory cell involvement in brain arteriovenous malformations.</a> Neurosurgery. 62, 1340-1349 (2008).</li><li>Martens, C. L., 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=Peptides+which+bind+to+E-selectin+and+block+neutrophil+adhesion.">Peptides which bind to E-selectin and block neutrophil adhesion.</a> J. Biol. Chem. 270, 21129-21136 (1995).</li></ol>

著者らは、開示することは何もありません。

成功した好中球/微小血管内皮細胞接着アッセイのための最も重要なステップは次のとおりである：1）低継数（&lt;9）、健康な内皮細胞の使用は、低密度（ すなわち &lt;5×10 6個の細胞で孤立した好中球を維持2） / ml）と分離の2時間以内にそれらを使用して、それぞれ、3）気難しいカルセインAMで標識された好中球の洗浄および好中球からのカルセインのECの汚染や損失を最小限...

血管内皮の循環血液と直接接触しているので、一意的に感染または損傷時に迅速な炎症反応を開始させる位置している。内皮細胞（ECS）の保存された細菌成分と危険分子の多様性、ならびにTNFαなどの炎症性メディエーターホストに対する受容体を認識する明白なパターン認識受容体。これらの受容体の活性化は、分泌性サイトカイン（ 例えば、IL-6、IL-8、CXCL1およびCCL2）へのECを誘導し、そしてその細胞表面1の接着分子（ 例えば、E / P-セレクチン、VCAM-1およびICAM-1）をアップレギュレートする、2。これらの分子は、すべての感染性病原体の宿主をクリアし、組織修復を3,4を開始するために感染や傷害部位への白血球の局在を促進する。感染に好中球応答が血管内皮と応答の間でよく調整された相互作用を伴う好中球。 EC活性化、ILアポン8が分泌され、感染症やけが5,6のサイトに家に好中球を可能に内皮に血管内の勾配を形成する。 E / P-セレクチン好中球の取り込みを仲介し、好中球の細胞表面上にglycomoleculesと比較的弱い団体を通じてローリング。これらの相互作用は、その同族受容体へのIL-8結合とともに、堅牢な、インテグリン媒介アタッチメントおよび内皮細胞表面7-10に好中球の最終的な逮捕を容易にします。停止した後、好中球は、直接病原体を排除するために、感染の特定の部位への脈管構造から移動病原体の拡散を防ぐために好中球細胞外トラップを生成し、創傷治癒を促進し、単球、マクロファージおよび樹状などの他の白血球を補充する追加の因子を放出することができ細胞11-17。 microvへの好中球の接着を定量するためのインビトロ方法は、この報告書に記載ascularはTNFα宿主炎症性メディエータによる活性化した後にECを。このアッセイは、ECの活性化のではなく、好中球を評価するために設計されている。初代ヒト好中球は、第密度勾配分離を用いて単離され、次いで、カルセインアセトキシメチル（AM）で標識される。生細胞加水カルセイン内エステラーゼは、492から495 nmおよび513から516 nmの18の放射の励起と高蛍光カルセイン分子にAM。蛍光標識された好中球は、その後、EC単層と共にインキュベートし、非付着性好中球は、その後、除去される。残りの結合した好中球の蛍光は、その後、蛍光分光光度計を用いて測定し、ウェルあたりの総好中球の蛍光入力のパーセントとして計算される。この方法は、分光光度法で使用される結合したカルセイン-標識された好中球を直接より定性的なEC交流から読み出さを与えるために蛍光顕微鏡を用いて可視化することができるというさらなる利点を有するtivati​​on。このアッセイは、静的条件下で行われるので、好中球の接着カスケードに生じるごく初期のイベントが評価される。これは、そのE-セレクチンとの相互作用が中断されたときTNFα処理ヒト肺微小血管EC（HMVEC-肺）単層への好中球の順守を大幅に低減されることを示すために、E-セレクチン遮断抗体を使用して、この報告書で確認された。 TNFαに加えて、我々は正常Toll様受容体1/2アゴニストペプチドグリカン関連リポタンパク質（PAL）、ムレインリポタンパク質（MLP）とPam3Cysによってヒト臍帯静脈EC（HUVEC）活性化の程度を決定するために、このアッセイを使用しているPam3Cys 19,20によってHMVEC-肺の活性化。さらに、我々は正常にこの方法論は、さまざまな互換性があることを示唆し、キナーゼ阻害剤とHMVEC-肺における表面および細胞質タンパク質のRNAiによるノックダウンした後に、このアッセイを使用した生化学的およびscreeninグラムアッセイ20。要約すると、このアッセイは、in vitroで炎症性メディエーターによってECの活性化の程度にアクセスするため、使いやすい、再現性、より機能的な方法を提供します。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>HMVEC-Lung</td>
 <td>Lonza</td>
 <td>CC-2527</td>
 <td></td>
 </tr>
 <tr>
 <td>EGM-2 MV</td>
 <td>Lonza</td>
 <td>CC-3202</td>
 <td></td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Life Technologies</td>
 <td>14175-095</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>48-well Tissue Culture Plates</td>
 <td>BD Falcon</td>
 <td>353078</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS (without phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL001 </td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>D-PBS (without Ca2+ and Mg2+ and phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL003</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>RPMI-1640 (without phenol red)</td>
 <td>Life Technologies</td>
 <td>11835-030</td>
 <td></td>
 </tr>
 <tr>
 <td>Polymorphprep</td>
 <td>Axis-Shield</td>
 <td>1114683</td>
 <td></td>
 </tr>
 <tr>
 <td>calcein AM</td>
 <td>Life Technologies</td>
 <td>C3099</td>
 <td>(0.995 mM) stock soln</td>
 </tr>
 <tr>
 <td>Trypan Blue Solution</td>
 <td>Sigma-Aldrich</td>
 <td>T8154</td>
 <td></td>
 </tr>
 <tr>
 <td>40 &mu;M filters</td>
 <td>VWR</td>
 <td>21008-949</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>FLUOstar OPTIMA Spectrophotometer</td>
 <td>BMG LABTECH</td>
 <td>-</td>
 <td></td>
 </tr>
 </tbody>
</table>

quantitative in vitro assay to measure neutrophil adhesion to activated primary human microvascular endothelial cells under static conditions

血管内皮は、炎症反応に不可欠な役割を果たしている。炎症の急性期の間に、内皮細胞（ECの）保存された微生物成分又は宿主由来の危険分子による宿主メディエーター直接によって活性化される。活性化されたECの特急サイトカイン、動員ケモカインおよび接着分子は、感染症やけがの部位で白血球を活性化し、維持する。好中球は到着する最初の白血球であり、両方の細胞の表面に存在する接着分子のさまざまな内皮に付着する。好中球の主な機能は、直接、微生物の脅威を除去するための追加の因子の放出を介して他の白血球の動員を促進し、創傷修復を開始することである。したがって、内皮への採用とアタッチメントは、炎症応答の開始に重要なステップです。本稿では、用いたin vitro好中球接着アッセイで説明カルセインAM-標識された一次ヒト好中球は、静的条件下で微小血管内皮細胞の活性化の程度を定量する。この方法は、蛍光分光光度計で定量し、同じサンプルは、直接結合好中球のより定性的評価のために蛍光顕微鏡を用いて可視化することができるというさらなる利点を有する。

好中球結合アッセイを使用して、信頼性、再現性のある結果を得るためには、 図1のHMVEC-肺に示すように、健康および微小血管内皮細胞のコンフルエントには、アッセイの当日に最適であることが不可欠である。また、低継番号微小血管のECは（9未満通路すなわち ）が使用されることが不可欠であり、従って、我々は、解凍から2週間以内にすべての実験を行うことをお勧?...

Watch this Scientific Journal Video about Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions at JoVE.com

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

感染部位における活性化内皮への好中球の順守は、ホストの炎症反応に不可欠なコンポーネントです。を可能にした好中球結合アッセイは、この報告書に記載<em&gt; in vitroで</em静的な条件下での炎症性メディエーターによって活性化内皮細胞への結合初代ヒト好中球の&gt;定量。

定量的な<em&gt;インビトロ</em静的な条件の下で活性化された初代ヒト微小血管内皮細胞への好中球の接着を測定するために&gt;アッセイ

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

quantitative-vitro-assay-to-measure-neutrophil-adhesion-to-activated

Research

JoVE Journal

Immunology and Infection

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

17.0K ビュー.  University of California, San Francisco. 感染部位における活性化内皮への好中球の順守は、ホストの炎症反応に不可欠なコンポーネントです。を可能にした好中球結合アッセイは、この報告書に記載 in vitroで定量。

ビデオ: Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

哺乳類上皮細胞上に細菌付着のインビトロアッセイ

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

免疫・感染学

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

フローの状況下で好中球移住研究におけるヒト臍帯静脈内皮細胞とそれらの使用の分離

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

不定冠詞<em&gt;のin vitro</em研究する&gt;共同感染モデル<em&gt;熱帯熱マラリア原虫（Plasmodium falciparum）</emヒトの第一次単球由来の免疫細胞に&gt;-HIV-1の相互作用

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

インビトロ 病原体誘導性好中球トランス上皮移動を評価するコーカルチャーアッセイ

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

せん断応力のヒト肝類洞内皮条件のことを白血球の募集を研究するフロー接着アッセイ

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

ヒト好フローチャンバー接着アッセイ

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

Tリンパ球におけるインテグリン活性化の研究のための静的な接着アッセイ

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

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 ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

in vitroおよびin vivoモデルでは血管壁の下でフローの状況に細菌付着を研究するために、

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Tリンパ球接着分子の相互作用の研究のための接着性の下でせん断応力の測定

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...