この研究は、ビクトリア州政府のOIS​​プログラム、国民健康、オーストラリアのプログラムやプロジェクトの助成金の医学研究評議会（JPFチン-塵）と大学院の奨学金（Dミッシェル）によって部分的にサポートされていました。

1。頸動脈を分離<ol><li> CO 2 / O 2窒息経由Euthanase10週齢のSprague Dawleyラット。 </li><li>最小限の血管の伸縮性を確保大動脈、心臓と消費税の左と右の総頸動脈。 </li><li>氷冷クレブス緩衝液中で大動脈と心臓から頚動脈を分離し、緊密な解剖を行う。 </li><li>マウントする前に氷上でクレブスで孤立した血管を保つ。 </li></ol>約。時間= 45分 2。プライミング容器室<ol><li> 37バッファを介し℃、carbogenガス（5％CO 2 95％O 2）を導入することで、生理的pHに維持血管チャンバーフラッシュクレブスバッファーの近位および遠位のコネクタで</li><li>チューブやカニューレが完全にフラッシュされると、整列されていることを確認します。 </li><li> P1（近位）とP2（遠位）変換器を介してクレブスバッファーをフラッシュします。トランスデューサには、気泡のないことを確認するためにタップを閉じます。 </li><li>対応するコネクタにトランスデューサを接続し、再び空気の泡を確実にない、よりクレブスバッファーをフラッシュします。チャンバーにタップをオフに閉じてください。 </li></ol>約。時間= 15分 3。血管の加圧室<ol><li>圧力機器の電源をオンにします。圧力サーボ、圧力モニター、蠕動ポンプ（図1）。 </li><li>圧力は20 mmHgの（1のダイヤル）でチューブを介して自動実行クレブス緩衝液の圧力のサーボで蠕動ポンプと、空気の泡を保証しません。 </li><li>閉じたP2トランスデューサにチューブを接続します。圧力が安定になります。 </li><li>どんな泡を吐き出すのに血管チャンバーにP2のタップを開くには、その後オフ閉じます。 </li><li>クレブスバッファー（ - 7 mLの5）と風呂を埋める。 </li></ol>約。時間= 10分 4。容器の取り付け<ol><li>各カニューレ上に解剖顕微鏡の代わりに黒のポリエステルネクタイ（図2）に基づく。 </li><li>離れてカニューレとカニューレホルダーを移動して、P1のカニューレ上にマウント¼容器（大動脈弓の端）。容器を引き裂くしないことを確認します。 </li><li>クレブス緩衝液で満たされた注射器を使用すると、静かにP1のトランスデューサーを介して血管の過剰な血液を洗い流す。チャンバーにP1をオフに閉じます。 </li><li>ポリエステルのネクタイとP1のカニューレに安全な容器。 </li><li> P2カニューレに取り付けるためのカニューレ/近いカニューレホルダーを移動します。 </li><li>遠位カニューレに容器の先端を（〜¼長さの）マウントします。ポリエステルタイで固定します。 </li></ol>約。時間= 20分 5。容器を加圧<ol><li>容器内に曲げたり、ストレッチを確保しないようにカニューレホルダーを調整します。 </li><li> P1とP2は、チャンバーに閉じた状態で自動から手動に圧力サーボを切り替えます。 10秒内の圧力の継続的な低下が存在しない圧力のサーボを確認してください。ある場合は、リークが発生している接続と変換器は、所定位置に固定する必要があります。 </li><li>チャンバーへの自動その後オープンP2に背圧サーボを調整します。顕微鏡下でタップをチャンバーに開いたときに血管が拡張させ観察。 </li><li>マニュアルに圧力のサーボを切り替えます。 10秒内の圧力の継続的な低下のためにもう一度確認してください。漏れがある場合、システムはタイトな圧力と容器内の穴になる可能性がありますされていません。 </li><li>チャンバーに自動オープンP1に切り替えます。手動設定で圧力が安定していることを確認してください。 </li><li>ない漏れの場合、手動設定の増加ダイヤルの2（40 mmHgの）へ。 </li><li>自動に調整し、顕微鏡下で血管を観察。必要に応じて、手動設定に漏れがないvessel.Checkには曲がりのないことを確認するためカニューレホルダーを調整する。 </li><li> 5.7所望の圧力に到達するまでに、3（60 mmHgの）を4（80 mmHgの）などのダイヤルを増やす - 手順5.6を繰り返します。 </li></ol>約。時間= 15〜30分 6。加圧容器をインキュベート<ol><li> 37温度コントローラセットを接続℃を</li><li>血管室バスへの2番目の蠕動ポンプ散らすクレブス緩衝液（1 mL /分）で。 </li><li>バスの唯一の最上位層が除去されることを保証室にアスピレータを接続します。 </li><li>自動に設定された圧力で1時間、37℃で加圧された容器をインキュベートする。 </li><li>圧力が定期的に（手動への切り替え）漏洩されていません確認してください。 </li><li>様々な薬理学的介入の効果は、単にインキュベーション中にお風呂に化合物を添加することによって観察することができます。 </li></ol>約。時間= 1時間 7。全血と加圧容器を灌流<ol><li>インキュベーション中に少なくとも7.5 mLのヒト全血/ 40 Uのヘパリン/ mLの血液中に収集された容器を得る。 37℃で50mlファルコンでインキュベート℃の</li><li> tの最後の10分前37彼10分間VybrantDil（1:1000）とインキュベーションのラベルの血·ダークのC。 </li><li> 10分後、気泡をクリアシリンジで血液を収集し、37℃で設定されたヒートジャケット℃でシリンジポンプに取り付けて</li><li>室/容器にP1のトランスデューサをオフに閉じ、シリンジと廃液チューブを接続する。 </li><li>任意の気泡を除去するため廃液チューブを介して1000μL/ minでパージの血液。 </li><li>チャンバーにP1を開きます。圧力のサーボは、所望の圧力を維持するために自動的に調整されます。 </li><li> 100μL/ minの血液を灌流。 </li><li>デジタルカメラのレコード1で15秒間灌流血管の2つのフィールド、3、5、7.5および10分に結合蛍光顕微鏡を使用する。 </li><li>郵便灌流血管内皮の完全性と機能がさらに炎症反応を検証するために免疫組織化学によって決定することができるようミオグラフと接着分子の発現などの薬理学的技術を介して評価することができる。 </li></ol>約。時間= 15分 8。代表的な結果： 圧力室のセットアップの概略図を図1に示されています。と蛍光顕微鏡の結果に結合デジタルカメラは、ライブビデオの録画を経由して瞬時に視覚化することができます。代表的なビデオ画像は、白血球が、彼 ​​らが10秒間静止残った場合内皮に付着すると考えられる図3に見られている。連続ループ接着細胞でのビデオ録画とフィールド当たりの平均としてカウントすることができます。低い（ 図3A）と高い（ 図3B）圧力の両方に密着性のある量の高い管腔内圧における白血球接着の顕著な増加を引き起こすだろうが見られ、これはまた、（ 図4）定量的に実証される。 <img alt="図1" src="/files/ftp_upload/3221/3221fig1.jpg" /> 図1。加圧ex vivoでの血管チャンバー概略図。近（P1）変換器と血圧が容器内で操作できるようにする、遠位（P2）変換器に接続されているカニューレ容器。灌流は、P1のトランスデューサーを介して、圧力はP2変換器を介して維持されます。 <img alt="図2" src="/files/ftp_upload/3221/3221fig2.jpg" /> 図2。タイでCannuala。黒いポリエステルのネクタイは、各カニューレに接続されています。 <img alt="図3" src="/files/ftp_upload/3221/3221fig3.jpg" /> 図3。灌流の10分後の代表的なビデオ画像を 80 mmHgの（）で蛍光下。ダイナミック細胞接着（赤矢印）、120 mmHgの（B）。 <img alt="図4" src="/files/ftp_upload/3221/3221fig4.jpg" /> 図4。低い（80 mmHgの）と高圧（120 mmHgの）で1時間のインキュベーション後のSprague Dawleyラット頸動脈の動脈における白血球接着。 *** P &lt;0.001、としてBonferroniの事後検定を用いて2方向反復測定ANOVAで分析した。

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治験実施計画書は、アルフレッド医学研究と教育分署動物倫理委員会とアルフレッド病院倫理委員会によって承認された。

これはリアルタイムで加圧条件下では無傷の孤立した血管の内皮への白血球の接着を研究するために修正された方法です。容器室の血流だけでは、大規模なマウスやラットの血管の炎症誘発性菌株の迅速な検証が可能になります。圧力操作を有効にすると、より良い模倣- ING生理学的および病態生理学的状態、動的な細胞間相互作用が非常に高い腔内圧力に低いから観察することができます。血管の直径は、十分な細胞イメージングプログラムを使用して測定することができるため、せん断流れおよび速度を決定し、したがって、操作することができます。 そのミオグラフ機能により、浴室に置かれた薬理学的介入は、このモデルは機械論的とシグナル伝達経路の研究を可能にすると可能な実験条件に別のディメンションを追加します。内皮保存が圧力の操作中に確認することはできませんが、アセチルコリンとPEへの応答がポスト灌流9を行うことができる。 それは、このセットアップは、細胞間の相互作用ではない拍動血流も収縮期または拡張期圧の影響についての管腔内圧の影響を示すことに注意する必要があります。白血球接着に対する急性圧力の変化が観察された一方さらに、、この設定はまた、慢性的な圧力の効果（すなわちインキュベーション時間の増加と慢性的な圧力の動物モデルを用いて）を見に利用することができます。のSprague Dawleyラット総頚動脈は、このセットにまで実証されていますが、他の系統と種は、カニューレのサイズに適切な調整で使用することができます。確かに、動物の年齢と体重がセットアップ船舶ごとに個別化する必要があることをこのように容器のサイズに影響を与えるとことに注意することが重要です。結合組織の密接かつ慎重に解剖は非常に白血球の可視化を向上させることができます。

<table border="1"><tbody><tr><th>試薬/装置の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td>顕微鏡</td><td>カールツァイス株式会社</td><td>ステレオディスカバリーV.20 </td><td></td></tr><tr><td> PHD 2000シリンジポンプ</td><td>ハーバード装置</td><td> 70から2016 </td><td></td></tr><tr><td>デジタルカメラとコントローラ</td><td>浜松ORCA - ER </td><td> C4742 - 95 </td><td></td></tr><tr><td>蛍光照明システム</td><td>ルーメンダイナミクス</td><td> X - Citeの120 </td><td></td></tr><tr><td>血管の商工会議所</td><td>リビングシステムの計装</td><td> CH/1/SH </td><td></td></tr><tr><td>圧力サーボコントローラとペリスタルティックポンプ</td><td>リビングシステムの計装</td><td> PS - 200 </td><td></td></tr><tr><td>灌流圧モニター</td><td>リビングシステムの計装</td><td> PM - 4 </td><td></td></tr><tr><td> 2 ×圧力変換器</td><td>リビングシステムの計装</td><td> PT - F </td><td></td></tr><tr><td>温度コントローラ</td><td>リビングシステムの計装</td><td> TC - 01 </td><td></td></tr><tr><td>蠕動ポンプ</td><td>インステックラボラトリーズ株式会社</td><td> P720 </td><td></td></tr><tr><td> VybrantDil細胞標識溶液</td><td>インビトロジェン</td><td> V - 22885 </td><td> 1:1000を使用してください</td></tr></tbody></table>

imaging leukocyte adhesion to the vascular endothelium at high intraluminal pressure

世界中で、高血圧は、人口の約4分の1であることが報告と死亡率、世界中の主要な生物医学的危険因子です。血管性高血圧では血管内皮機能障害とアテローム性動脈硬化症や慢性腎臓病2、脳卒中3心不全4など様々な疾病状態につながる増加炎症に関連付けられています。アテローム発生につながる血管の炎症の初期段階では、内皮を介して白血球のローリング、テザリング、アドヒアランスやその後の転生を含む接着カスケードです。内皮への白血球の動員と蓄積は、血管細胞接着分子-1（VCAM - 1）、細胞内の細胞接着分子-1（ICAM - 1）とE -セレクチンと同様の増加などの接着分子の発現増加によって媒介されるサイトカインとケモカイン放出と活性酸素種のアップレギュレーション5。このような静的な接着アッセイなどのin vitro法では細胞間接着に関与するメカニズムだけでなく、細胞接着分子の解析を判断するのに役立ちます。 in vitro試験前で採用されている手法は、内皮上に圧力の急性の増加が単球接着、6しかし、in vitro試験の多くと同様に、これらの結果がリアルタイムで行われていない接着分子と炎症マーカーの発現上昇につながることができることが実証されている生理的流れの条件下で、また全血を持つ。したがって、in vivo試験では 、ますます血管炎症とプラークの開発を実証するために動物モデルで利用されています。生体顕微鏡検査は現在では広く白血球接着、ローリング、マイグレーションと転生7-9を評価するために使用されます。 in vivo試験内皮接着する白血球に圧力の効果を組み合わせるときには、それほど広範です。そのような研究は、免疫組織化学10を介して評価された動脈の成長とリモデリングが炎症マーカーのフローとせん断のリアルタイムエフェクトを調べます。ここでは、全血灌流を使用して無傷の加圧された血管のリアルタイムで白血球の接着を記録するためのモデルを提示する。方法論は、無傷の血管の白血球-内皮接着剤の相互作用のリアルタイム解析を可能にするex vivoの血管室灌流モデル9の変更です。私たちの修正は、管腔内圧だけでなく、生理的な流れの条件だけでなく、加圧条件下での試験を可能にする最大200 mmHgの操作が可能になります。圧力の筋運動記録法のシステムが以前に血管壁と内腔直径11だけでなく、血管の収縮を観察することが立証されているが、これはリアルタイムで白血球-内皮相互作用を示す初めてのことです。ここでは、頸動脈ラットから採取し、蛍光顕微鏡に結合カスタムメイドのフローチャンバーへのカニューレを使用して技術を実証する。容器のチャンバーは、画像の容器を、短い作動距離と大口径対物レンズを可能にする大規模な底のカバーガラスが装備されています。さらに、選択されたアゴニストおよび/またはアンタゴニストは、さらに細胞接着を制御するメカニズムを調べるために利用することができます。生体顕微鏡に比べてこの方法の利点は、侵襲手術のない関与を含んでいないため、より高いスループットを得ることができます。生体内では唯一の全身阻害剤の治療を可能にするのに対し、この方法はまた、所望の容器にローカライズされた阻害剤の治療を使用できるようになります。

Watch this Scientific Journal Video about Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure at JoVE.com

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

これは収穫加圧された血管の内皮への白血球の接着を可視化する方法です。テクニックは、このように高血圧の病態生理学的条件を模倣 - イング200 mmHgに管内の圧力に異なるとのせん断流動下で血管の密着性を研究することができます。

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. ...

imaging-leukocyte-adhesion-to-vascular-endothelium-at-high

Research

JoVE Journal

Immunology and Infection

15.5K ビュー.  Monash University. これは収穫加圧された血管の内皮への白血球の接着を可視化する方法です。テクニックは、このように高血圧の病態生理学的条件を模倣 - イング200 mmHgに管内の圧力に異なるとのせん断流動下で血管の密着性を研究することができます。

ビデオ: Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白血球ホーミングと移行の非侵襲的イメージング in vivoで</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

免疫・感染学

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

全葉のイメージングを使用して細菌細胞をライブに気孔反応を評価する

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

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 ...

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 ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

ファージディスプレイされた合成抗体ライブラリーから、スケーラブルな高スループットの選択

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

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 ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

マウスの腸間膜静脈内の生体顕微鏡白血球 - 内皮及び血小板 - 白血球の相互作用

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

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 ...