This work was supported by EMBO (to Y.E.), Foundation Machaon (to Y.E.) and SNSF (to B.A.I.). We thank the Bioimaging Core Facility for the availability of the Nikon A1r microscope and technical assistance. We thank Mrs. Clarissa Bartley for English correction.

注：動物の手順は、スイスのジュネーブで動物ケアの機関倫理委員会、州立獣医事務所に基づいて行われました。認証番号GE / 14分の63。 骨髄からの単一細胞懸濁液の調製<ol><li>頸椎脱臼により犠牲マウス（8〜12週齢）。 70％エタノールで後ろ足を滅菌します。後ろ足から肌を削除します。 </li><li>マウスの大腿骨と脛骨を解剖し、メスで脚から組織を除去します。 PBSで満たされた培養皿に90％のエタノールと場所で足を洗うこと。 </li><li>膝関節でメスと独立と大腿骨と脛骨から培養フード、クリーンな組織の下で。骨の両端をカット。 </li><li> 23G針および調製培地を充填した1mlシリンジ（フェノールレッドを含まないDMEM / F12、1％ラット血清）を用いて、各骨からフラッシュ骨髄、50 mlのチューブにねじ。微妙23G NEを通じて継代することにより、細胞凝集体を混乱させるedleと1mlシリンジ。準備中で25ミリリットルに全量を。 </li><li>遠心分離機250×gで、5分間、4°C。上清を捨てます。 1ml中に再懸濁ペレットを赤血球溶解RBLCバッファー15ml中のチューブをネジ（8.3グラムのNH 4 Clを 、蒸留水で1リットルに完全に1グラムのNaHCO 3、1ミリリットルのEDTA（100 mM）の、0.22μmのフィルタ処理）。氷上で30秒インキュベートします。 </li><li>準備中と遠心分離250×gでの10ミリリットルを追加し、5分、4℃。 2ミリリットルの調製培地中の上清と再懸濁を捨てます。 </li></ol>ネガティブ選択により2好中球濃縮<ol><li>マウスの好中球濃縮カクテル（細胞単離プラットフォームキットなどEasySepなど）を150μlを追加します。混合し、氷上で10分間インキュベートします。 </li><li>フェノールレッドを含まないDMEM 10mlのを追加します。一度裏返し、遠心分離機250×gで、3分、4°C。 </li><li>上清を捨てます。下部チューブラウンド5ミリリットルポリスチレン中1.75ミリリットル準備中で再懸濁ペレット。 250μlのビオを追加選択カクテルインチ混合し、氷上で10分間インキュベートします。 </li><li>粒子を再懸濁するために30秒間（細胞単離プラットフォームキットから）磁性粒子を渦。細胞懸濁液に500μlの磁性粒子を追加します。混合し、氷上で10分間インキュベートします。 </li><li>磁石にチューブを置きます。 3分を待ちます。 </li><li>目的の非標識細胞を収集するために、下部のチューブ新ラウンド5ミリリットルポリスチレン上に磁石を反転。チューブにぶら下がって最後の一滴を取ってはいけません。不要な細胞は、磁石内部チューブ内に残ります。バイオハザード廃棄物の中に、このチューブを捨てます。 </li><li>磁石に新しいチューブを置きます。 3分を待ちます。 </li><li>新しい15mlを希望する非標識細胞を収集するためにスクリュー管に磁石を反転。チューブにぶら下がって最後の一滴を取ってはいけません。 </li><li>フェノールレッドを含まないDMEMで5ミリリットルへの完全なボリューム。 </li></ol>好中球の3ラベリング<ol><li>このような細胞トラッカーオレンジ（使用濃度としてオレンジ色の色素の0.5μLを追加1μM、CMRA - クロロ - rodol酢酸、DMSO中の10mMストック）。 37℃で2分間インキュベートします。 </li><li>準備中の2.5ミリリットルを追加します。遠心分離機250×gで、5分間、4°C。 </li><li>上清を捨てます。 1.5mlチューブで1ミリリットル準備中で再懸濁ペレット。 </li><li>血球計数器でカウントするアリコートを取ります。 </li><li> 37℃で5分間インキュベートします。遠心分離機250×gで、5分間、4°C。 </li><li>上清を捨てます。 1mlのPBS中に再懸濁ペレット。遠心250×gで5分間、洗浄するために4℃。 </li><li>上清を捨てます。 100μlのPBSあたり10×10 6個の細胞として再懸濁ペレット。静脈内注射するまで氷上で保管してください。注：細胞は、可能な限り迅速に静脈注射する必要があります。 6-12x10 6好中球/マウスは、通常、得られます。 </li></ol>生体顕微鏡4.マウスの準備注：ステップ4.1）および4.2）は、IC上の標識された好中球の延長待ち時間を回避するために、実験の開始時に実行されるべきですE。 <ol><li> （8-12週齢）CX 3 CR1 GFPマウスを計量。 </li><li> （PBS中1.75ミリグラム/キログラムacepromazoneケタミン60 mgの/ kgで、キシラジン4.5mgの/キログラム）麻酔薬の800μLを準備します。 </li><li>マウスを麻酔。マウスを200μl/ 20gで射出IP。ピンチと呼吸数をチェックすることにより、TOEによって制御麻酔。 </li><li>静脈内に実験者の都合で、外側尾静脈または眼窩洞を使用して、好中球（100μlのPBS中の10×10 6細胞 ）を注入します。 </li><li>加熱パッドの上にマウスを置くと、眼のゲルで目を保護します。 </li><li>ガラスカバースリップ（直径35mm）の直径30mmの穴を有する10cmの組織培養皿に入れます。使用する油は、カバーガラスと接触している組織培養皿を維持することです。 </li><li>腹膜を露出するようにはさみで腹部の皮膚を切開。腸を露出するようにハサミで腹膜を切開。 </li><li> Pに200μlのPBSを（37℃に予熱）を追加しますeritoneal空洞。あなたの手でマウスを取り、腸は穏やかな圧力で腹腔から外れているので、それは、下向きに回します。組織培養皿にマウスを置きます。 </li><li>静かに腸間膜の血管を露出させるためにカバースリップ上に滅菌綿棒で腸をderoll。 </li><li>バンドにティッシュペーパーをカットし、37℃で加熱し、PBSでそれらを濡らし。蠕動運動から生じる運動を減少させるためにティッシュペーパーの作品で腸を固定。 </li><li>倒立顕微鏡のカスタムメイドのトレイステージに、37℃に温度調節チャンバー内に組織培養皿やマウスを置きます。後肢に麻酔薬SCを含む注射器を導入。注：また、マウスマスクで酸素（0.5 L /分）を受信することができます。 </li><li>つま先をつまんや呼吸数を30分毎にチェックすることによって、麻酔のための制御マウス。必要に応じて、適切に麻酔を維持するために、麻酔薬のブースト（20μl）を提供します。 NOTE：血管系の乾燥は避けるべきです。したがって、その湿度を定期的に37°Cに加温したPBS腸を固定するために使用される紙組織を湿潤することによって維持されます。 </li></ol> 5.生体顕微鏡を用いてin vivoで炎症を起こした船舶に好中球および単球挙動を測定<ol><li>レーザー誘起光毒性を避けるために必要な最低限の電力で488と561-にレーザーを設定します。我々の実験では、0.5％未満です。好中球、単球におけるGFPとオレンジ染料の強度は、通常、0.3％が十分であるように明るいです。注：これは、我々のシステムで0.15 mWの0.25へのレーザーパワーを表します。 </li><li>倒立顕微鏡の20X乾燥対物レンズを使用して開いたピンホールで2.2秒で512×512ピクセル（ すなわち、635ミクロン）の画像を取得します。低レーザ出力で十分な信号を確実に、10〜20μmの間にZ深度を使用してください。 注：我々は、通常12〜16の間のZ深度で実験を行います56;メートル。発表の数字や映画の場合には、Z深度は20μmです。位相差は、内皮壁の同定を可能にし、イメージングは​​腸間膜静脈に行われます。パトロール細胞が関心のある分野を記録し、5〜10ミクロン/分6の速度で、非常にゆっくりと移動するので、すべての20秒で十分です。記載されている設定では、電動ステージが同時に関心を最大7つのフィールドを記録することができます。 </li><li>彼らは最初の映画の獲得30分前の準備から回復することを可能にしているマウスと顕微鏡の恒温槽内の腸間膜血管を置きます。この間、関心のいくつかのフィールドを選択します。客観的に最も近い血管の管腔表面に焦点を当てています。必要に応じて、フォーカスを設定する内皮のわずかな自家蛍光を利用します。 注：マウスの​​準備中に手術は少し内皮を強調しています。その結果、好中球のローリングと/または単球をマウスの準備の後の最初の15分間で観察することができます。 </li><li>定常状態の条件の下で30分、1画像ごとに20秒間の最初の映画を記録します。 注：ソフトウェアが2.2秒の関心の各フィールドを取得し、電動ステージが自動的に他の1つのフィールドから移動します。なお、記録映画の30分の間に戻って20秒以内に最初の位置に戻ります。 </li><li>血管の炎症を開始するには、画像化された血管に直接TLR2 / TLR1アゴニストPam3CSK4 12を100μgを含む20μlのPBSのドロップを追加します。 </li><li>興味のある各フィールドのフォーカスを制御します。 注：取得の過程で、z軸上の焦点のわずかな変化が常に存在します。 10-20ミクロンのZ深さにもかかわらず、これは、蛍光強度の減少をもたらすことができます。必要に応じてそのため、手動で、各ムービーの最後に関心の各フィールドを再び焦点を合わせます。 30分-期間についてとに3時間まで記録映画TAL炎症の開始後。 </li><li>実験の最後に、頚椎脱臼により麻酔したマウスを生け贄に捧げます。 </li></ol>白血球の動画ファイルと追跡の6世代注：ニコン収集ソフトウェアが.nd2ファイルを生成しますが、次の手順では、他のソフトウェアとブランドと同様です。 <ol><li> TIFFシリーズとしてエクスポートファイル。 </li><li> ImageJソフトウェア（に行く<a href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ij/）。</a> TIFFシリーズをインポートします。別のファイルとして各チャネル。 </li><li>バックグラウンドノイズを低減するために、緑と赤のチャンネル（処理&gt;フィルタ&gt;中央...）2.0ピクセルの半径のメディアンフィルタを適用します。 </li><li> （バイナリを作る&gt;プロセス&gt;バイナリ）のバイナリにファイルを変換します。各画像のしきい値を計算するためのソフトウェアを有効にします。 </li><li>一つの画像シリーズ（画像&gt;カラー&gt; ...チャンネルをマージ）にチャンネルをマージします。 GREEで単球を選択nチャネル、赤チャネルと灰色のチャネルでの透過光中の好中球。 </li><li> RGBの各色に積み重ねられた画像を変換する（画像&gt;カラー&gt; RGBにスタック）。 </li><li> .AVIなどのファイルを保存します</li><li>データがインポートされ、IMARISソフトウェア（ビットプレーン）にまとめられています。単球および好中球の運動はその後、スポットトラッキングウィザードを使用して追跡されます。 </li></ol>

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The authors declare that they have no competing financial interests.

この原稿に記載された方法論は、効率的に定常状態および炎症状態下腸間膜静脈に単球および好中球の挙動を研究するための一貫したアプローチを提供します。 準備の重要なステップは、PBSで湿っ組織と腸の固定化です。正しく実行した場合、腸間膜血管がうまく画像取得のためのカバースリップ上に露出されています。これは、腸間膜静脈の白血球の挙動を監視する?...

<html>好中球および単球は、継続的に血液中を循環自然免疫系の細胞です。傷害または感染すると、炎症性シグナルは、ここで、細胞性免疫応答1-3を開始し 、破損して感染した組織への白血球の漏出を誘導します。白血球動員の迅速性は、免疫応答の肯定的な結果を決定します。これらの複雑なプロセスは、白血球に関与する分子の洞察を得る.TO循環白血球と内皮1-3との間の接着の連絡先を確立するために役立つ炎症を起こした内皮に存在する特定の分子（例えば、セレクチン、内皮に結合したケモカイン）に依存しています採用カスケードは、細胞動員の動態を可視化するために、各セル/集団の挙動を追跡することが重要です。効果的な方法は、目的の細胞内で蛍光タンパク質を発現するトランスジェニックマウスに生体顕微鏡を含みます。 現在までの内容は&quot;&gt;、生体顕微鏡を用いて、いくつかの方法が画像に開発された血管系4,5。例えば、耳の真皮の血管系や生体共焦点顕微鏡による腸間膜静脈の撮像は、マウスLy6C 低い単球および人間のパトロール動作を識別するために使用されましたCD14は、定常状態条件6~8の下の血管の管腔表面上のCD16 +単球を暗く。マウス精巣挙血管系モデルは、多くの場合、トランスジェニックマウスにおける炎症性または虚血状態下での好中球または炎症Ly6C 高い単球の挙動を監視するために使用される。それは場合、精巣挙がIL1β、CCL2、TNFβまたはfMLPのの陰嚢内注射を介して刺激される。2-4時間後、組織はその後、外科的に露出されており、生体共焦点顕微鏡9-11で分析しました。 以下のプロトコルは、任意のと同時に、単球および好中球を監視するための方法を記載倒立蛍光共焦点顕微鏡。また、私たちの方法は、白血球動員の動態を追跡するためにどのように前の画像と同じ容器を（定常状態）にし、炎症後及び方法について説明します。この目的のために、我々は、その単球のeGFPを発現し、IVオレンジ色素で標識されたマウスの好中球を注射されたCX 3 CR1 GFP /重量マウスを使用しています。倒立共焦点顕微鏡を使用して、（1）追跡し、定常状態と、（2）局所炎症後の同じ容器の両方の単球および好中球の動員を追跡する下パトロールLy6C 低い単球を分析することが可能です。ここでは、Pam3CSK4 12 TLR2 / TLR1アゴニストを使用して炎症状態を作成します。また、このような撮像は、特定のノックアウトマウスまたは遮断抗体6,9,13の存在下で実行した場合の白血球動員カスケードの様々な段階での関心の特定の分子の役割を決定するのに役立つことがあります。

<table>
	<tbody>
		<tr>
			<td>5 mL polystyrene round bottom tubes&#160;</td>
			<td>Beckton Dickinson</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>10x CFI Plan Apochromat 0.45&#160;&#160; DT:4mm&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20x CFI Plan Apochromat VC 0.75&#160;&#160; DT:1mm&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Tracker Orange CMRA Dye</td>
			<td>Life Technologies</td>
			<td>C34551</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>Stem Cell Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Mouse Neutrophil Enrichement kit</td>
			<td>Stem Cell Technologies</td>
			<td>19762</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>PAA</td>
			<td>A15-042</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion Oil Type A</td>
			<td>Nikon</td>
			<td></td>
			<td>any viscous oil&#160;</td>
		</tr>
		<tr>
			<td>Life Box Temperature Control System</td>
			<td>Life Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sigma Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R confocal microscope</td>
			<td>Nikon</td>
			<td>A1R</td>
			<td>inverted microscope, motorized x/y/z stage, NIS elements software</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>phenol red free DMEM/F12</td>
			<td>Life Technologies</td>
			<td>21041-025</td>
			<td>any phenol red free medium is suitable</td>
		</tr>
		<tr>
			<td>PAM3CSK4</td>
			<td>Invivogen</td>
			<td>tlrl-pms</td>
			<td>reconstitute in PBS</td>
		</tr>
		<tr>
			<td>Rat serum</td>
			<td>Stem Cell Technologies</td>
			<td></td>
			<td>included in EasySep Mouse Neutrophil Enrichement kit</td>
		</tr>
		<tr>
			<td>tissue culture dish 100</td>
			<td>TPP</td>
			<td>93100</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging neutrophils and monocytes in mesenteric veins by intravital microscopy on anaesthetized mice in real time

効率的な免疫応答は、感染または傷害の部位への血液の白血球の急速な動員に依存しています。 in vivoでの白血球遊走を調査することは、血管内皮と白血球の経内皮遊走との相互作用の分子基盤を理解するために重要です。一つの強力なアプローチは、目的の細胞に蛍光タンパク質を発現するトランスジェニックマウスの生体顕微鏡を必要とします。 ここでは、倒立型共焦点顕微鏡でオレンジ色の色素で標識された好中球を注射されたCX 3 CR1 GFP /重量マウス静脈撮像単球および好中球のためのプロトコルを提示します。タイムラプスムービーが両方の定常状態および炎症状態下腸間膜静脈の白血球の挙動の分析を可能にする画像の数時間に30分から集められました。ローカルTLR2 / TLR1アゴニストPam3SK4と血管の炎症を誘導し、subsequeを監視するために、我々はまた、手順を説明します好中球および単球のNT募集。 提示された技術はまた、白血球の他の集団を監視し、他の刺激またはトランスジェニックマウスを用いて白血球動員または輸送に関与する分子を調査するために使用することができます。

原稿を簡単にリアルタイムで麻酔したマウスの腸間膜静脈内単球および好中球の挙動を監視するために最適化されたプロトコルを記述します。 37°C-サーモスタット室の使用は、マウスの温度を維持し、また、白血球の温度依存移動によることが必須です。マウスの作製は、 図1、図2に示す顕微鏡で見たすべての領域に表示されます。透過光は、腸間膜静脈（赤矢印）と動脈（青?...

Watch this Scientific Journal Video about Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time at JoVE.com

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

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 ...

imaging-neutrophils-monocytes-mesenteric-veins-intravital-microscopy

Research

JoVE Journal

Immunology and Infection

10.4K ビュー.  University of Geneva. 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.

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

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

生体内ビデオ顕微鏡による組織内好中球腔内クロールを追跡、経内皮移動と走

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

免疫・感染学

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 ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

の相互作用を研究<em&gt;黄色ブドウ球菌</em&gt;フローサイトメトリーとタイムラプス顕微鏡による好中球と

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

胚中心の高解像度生体内ストライプ照明顕微鏡

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

における骨髄細胞ダイナミクスのリアルタイムイメージング<em&gt;のApc<sup&gt;最小/ +</sup</em&gt;腸腫瘍ディスク共焦点顕微鏡スピニングすることにより

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

CXCR6.Gfpレポーターマウスを使用して、健康で罹患肝臓における免疫細胞の長期生体内多光子顕微鏡イメージング

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

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 ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

に応答したヒト一次好中球及び単球による抗カビ活動のライブイメージング<em&gt; A。フミガーツス</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

イメージング 2 光子生体顕微鏡を用いたインフルエンザ ウイルス感染症の中に気管粘膜の細胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

インビタル顕微鏡で評価されたマウスにおけるクレマスター筋の白血球浸潤

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...