The authors thank the Central Animal facility of the University Hospital Aachen for technical support. This work was supported by the German Research Foundation (DFG Ta434/2-1, DFG SFB/TRR 57) and by the Interdisciplinary Center for Clinical Research (IZKF) Aachen. This work was further supported by the Core Facility &#8221;Two-Photon Imaging&#8221;, a Core Facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University.

注：実験は、動物の保護に関する（NIH出版、第8版、2011年）「実験動物の管理と使用に関する指針」及び指令63分の2010 / EU以下の動物実験を支配するドイツの法律に従って行った科学的な目的（欧州連合官報、2010）に使用される。公式許可は政府の動物の管理と使用オフィス（LANUVノルトラインヴェストファーレン、レックリングハウゼン、ドイツ）から付与されました。 注：短期画像化のために省略することができる手順の準備時間を削減し、外科的プロトコルを簡略化するアスタリスク（*）が付いています（ 例もショット、3-Dスタックまたは短期間のタイムラプス顕微鏡をスナップ）。必要に応じて、しかし、生存期間が顕著に減少する場合の撮像には、広範囲の監視および制御を循環することなく行うことができる。 1.顕微鏡のセットアップと手術前の準備（5-10マイルN） <ol><li> （顕微鏡、パワーラボ、レーザー、加熱パッド、気象室、Webカム、シリンジポンプ装置、人工呼吸器）上のスイッチシステム。 </li><li>イソフルラン蒸気と設定イソフルランレベルへのO 2供給が1.5体積％に接続します（V / V）、小動物人工呼吸器に接続します。 <ol><li>短期間のイメージングのために、唯一の初期腹腔内麻酔誘導後、イソフルランを用いて麻酔を維持し（ステップ2.2参照）。次いで、安定な肝臓微小循環を保証するために2巻の％（v / v）のイソフルランを使用して、2時間未満に撮像時間を維持する。 </li></ol></li><li> 150〜200μLの容量で120〜140呼吸サイクル/分に呼吸を設定します。 </li><li>マウスのアガロース段階を設定します。 40°の角度でディッシュ（約10cm×15センチベース）に注ぐこと、3％（w / v）アガロース100mlのを準備する。 </li><li>必要な機器を収集外科作業領域を設定します。微生物感染を防ぐために、w / vのSekuseptフォルテSの1％溶液（9.9％を使用して器具を消毒する5分です。v、ホルムアルデヒド/ w）の、グルタラール9.8％実験（ 図1A）の前に。 </li><li>残りの成分を得る：皮膚消毒剤（ 例えば 、10％ポビドンヨード溶液）、肝臓ステージ（スタック·ブリッジ）、アガロース溶液（3％（w / v）のPBS中の）5％、シリンジポンプ（w / v）のグルコース溶液を麻酔液I（ケタミン0.1mg / mlの、キシラジンを0.5mg / mlのフェンタニル5μg/ ml）を、綿棒、n-ブチル-2- Cyanoacrylat、及び1×PBSで（G-5）、シリンジポンプ。予熱のインキュベーション室にアガロース段階ディッシュを入れてください。 </li></ol> 2.気管切開（10〜15分） <ol><li> 25〜28グラムの重さ、家の繁殖でからC57BL / 6マウスを使用してください。 12時間明/ 12時間暗サイクルで、温度と湿度が管理された環境でのハウスFELASAガイドラインに従って特定病原体フリーの条件下ではマウス、。動物に標準マウス食餌（スニフ標準的なげっ歯類食）と水を自由に無料でアクセスを許可します。 </li><li> initによってマウスを麻酔IAL腹腔内麻酔液II（ケタミン0.1 mg / mlの、キシラジン1 mg / mlの、ブプレノルフィン10μg/ ml）を250μlの内（ip）注射。 <ol><li> *生体内撮像中維持費のために、継続的に連続的な腹腔内注射により麻酔液Iを投与し（ケタミン0.1mg / mlの、キシラジンを0.5mg / mlのフェンタニル5μg/ ml）を0.2ミリリットル/時間の流量でシリンジポンプ装置を使用して吸入イソフルラン0.5巻の％との組合せ（v / v）である。 </li><li> 5分後の反射神経をチェックします（ 例えば鉗子でフットパッドピンチ）、気管切開のために皮膚を消毒、マウスは外科トレラント麻酔状態にある場合の準備を開始。 </li><li> （ 図1B）を乾燥から角膜を防ぐために、目の保護クリーム（ 例えば 、デクスパンテノールは50mg / g）を適用します。 </li></ol></li><li>四肢のために粘着テープを使用して腹側を露出する準備テーブルの上にマウスを固定。穏やかに、この位置に固定する、例えば 、首を引き伸ばし、ゴムバンドを使用して、tはフック切歯O。 <ol><li>綿棒で消毒剤を適用することにより、ポビドンヨード溶液を用いて、皮膚や毛を消毒する。 </li></ol></li><li>直接顎の下に、最初の皮膚カット（0.5〜長さ1cm）を実行する（ 図1C） <ol><li>慎重に唾液腺（ 図1D）との間に結合組織を解剖。 </li><li>慎重に開いている筋肉の管周囲の気管（ 図1E、F）を引き裂く。 </li></ol></li><li>後で換気チューブの固定（ 図1G）のための気管の下に外科スレッドを置きます。 <ol><li> T字型の切開を行う軟骨リング間のマイクロハサミでオープン気管（ 図1H） </li></ol></li><li>切開（〜0.5センチメートル）を介して気管内に換気チューブを置きます。 、前方のチューブをプッシュ小さな解剖鉗子で尾方端をつかみ、ゆっくりと気管にチューブを前進させるために（ 図1I） </li><li>外科糸でチューブを固定（ 例： </e気管内部の管の周囲結び目を結ぶステップ2.5に配置されたスレッドとM&gt;、5-0）;偶発depositioning（ 図1J、K）を避けるために、皮膚の管の第2頭蓋固定を行う。 </li><li>シールは、組織接着剤（ 例えば 、n-ブチル-2- Cyanoacrylat）（ 図1L）で切断した。 </li><li>粘着テープを使用して先頭にチューブを固定する。 </li></ol> 3.開腹（15〜20分） <ol><li>低体温症を防ぐために加熱パッド上にマウスを置きます。腹部を剃る、慎重に皮膚から毛を取り除く。ポビドンヨード溶液を用いて剃毛した皮膚や毛を消毒。 </li><li>外科用ハサミ（ 図2A）を使用して、胸骨の下にある小さな皮膚カットを行います。出血を防ぐために、すべての可視血管を焼灼、両側に横方向に肋骨の下にカットを拡張します。 </li><li>慎重に腹膜（ 図2B）を開く、胸骨下の白線の小さなカットを行う。 cauterizを使用して両側にカットを拡張ationが（ 図2C、D）を出血を防ぐために。 </li><li>アガロース段階皿（ 図2E）にマウスを置きます。マウス（通常は12〜14カバースリップ）（ 図2F）の両側に適切な高さのスタックを置きます。 </li><li>呼吸に顕微鏡を同期させるために背中の上部の下に呼吸トリガーセンサを配置します。トリガーユニット（ 図2G）をアクティブにします。 </li><li>リブ（ 図2I）を後退させる胸骨を通して外科糸（5-0）を配置します。 </li><li>慎重に湾曲した外科用ハサミを使用して、肝臓および横隔膜（鎌状靱帯）、ならびに肝臓および胃腸管を連結する靭帯を切断する。大動脈（ 図2J）まで鎌状靭帯をカット。 </li></ol> 4.サンプルのセットアップ（10〜15分） <ol><li>大型肝葉へ簡単にアクセスするための45°の角度で右側にマウスを置きます。 </li><li>腹部をカバーし、肋骨下のステージングブリッジを追加します。空洞。鋭いエッジ（ 図2K）を覆うように粘着テープ被覆を有する標準カバースリップを使用してブリッジを製造する。 </li><li>ステージ上で大規模な肝葉を置きます。慎重にダブルボールスタイラスプローブまたは肝臓下のパッド入りのへらをスリップし、湿った綿棒やウェットティッシュを使用して臓器の上部を保持する。スライド上に葉を持ち上げて、穏やかなドラッグを適用します。ローブは曲げたり折り畳むことができる。肝臓（ 図2L）の唯一の穏やかな操作に余分なケアを提供する。 </li><li> 例えば 、小さなカバースリップ（20ミリメートル×20ミリメートル）、その横に肝葉のほぼ同じ高さの山がカバースリップをサポートするために、横方向のサポートステークスを、置きます。 </li><li>肝葉に大きなカバースリップ（24ミリメートル×50ミリメートル）を配置します。そのカバースリップは、（ 図2M）できるだけ水平に配向されていることを確認してください。 注：カバースリップは、それを圧迫することなく、組織と接触しているべきである。血流障害（白組織）の目に見える兆候を確認してください。 microcirc場合、ulationが破壊され、さらに支援する株式を追加します。 </li><li> *長期の麻酔およびG-5アプリケーションのために二つの独立した腹腔内カテーテル（ 図2N）を配置します。次の後肢に下腹部に横方向にカテーテルをインストールします。 注：腹腔カテーテルは柔 ​​軟なシリコーンチューブ（ 図3）27 G針を接続して自作することができる。 </li><li> *皮膚に5-0外科用糸のループを有する固定する針が偶発的変位を回避する。 </li><li> * I 1カテーテルする麻酔溶液とシリンジポンプを取り付け、0.2ミリリットル/時間に設定された流量、カテーテル2にG-5とシリンジポンプを取り付け、0.1ミリリットル/時に流量設定。 </li></ol> 5.マウスの監視<ol><li> *場所のECGフロントに電極および後肢四肢（ 図4A）。 </li><li> * 2レベルセンサをCOに有効期限のチューブを取り付けます。 </li><li> （ 図4Cの熱パッドに接続された外部温度センサを追加する）。 </li></ol> 6.埋め込み、組織固定（5〜10分） <ol><li> 1X PBSにアガロース（w / v）の3％の100ミリリットルを準備します。温度を5 mlシリンジと18 G針（ 図5A）を用いて41°Cである場合に肝臓を埋め込 ​​む。 </li><li> （ 図5B、C）カバースリップ上で、マウスの周りに残っているアガロースを注ぐ。アガロースが完全に糊化するまでお待ちください。 </li><li>準備肝葉（ 図5D、E）をスキャンするために十分なviewfield大型の準備、ハイデマンのへらを使用して余分なアガロースを削除します。 </li></ol> 7.イメージング<ol><li>顕微鏡気候室にマウスを転送します。 </li><li> 1×PBS（37℃に予熱した）の50〜100ミリリットルを追加します。 </li><li>蒸発（ 図5F）を防止するために試料皿をカバーしています。 </li><li> * 0.5体積％（V / V）に吸入イソフルランを減らします。 </li><li> *、ソフトウェアを監視するECG、心拍数及び呼気CO 2の記録を開始。 </li><li>イメージングを起動します。オープンラのSerシャッター、Z-スタックの上限と下限を定義し、関心のビューのフィールドを識別し、時間経過の記録を開始。必要に応じて、Z-ドリフト（ 例えば 、血液の圧力または温度変動の変化に、 図（5G）を補正するために、Zスタックを再調整する。 注：撮像期間終了後、または動物の循環や麻酔が不安定になった場合、（麻酔からの覚醒せず）頸椎脱臼によりマウスを生け贄に捧げる。 </li></ol>

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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=Macrophage+and+T+cell+dynamics+during+the+development+and+disintegration+of+mycobacterial+granulomas.">Macrophage and T cell dynamics during the development and disintegration of mycobacterial granulomas.</a> Immunity. 28 (2), 271-284 (2008).</li><li>Beattie, 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=Leishmania+donovani-induced+expression+of+signal+regulatory+protein+alpha+on+Kupffer+cells+enhances+hepatic+invariant+NKT-cell+activation.">Leishmania donovani-induced expression of signal regulatory protein alpha on Kupffer cells enhances hepatic invariant NKT-cell activation.</a> Eur J Immunol. 40 (1), 117-123 (2010).</li><li>Beattie, 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=Dynamic+imaging+of+experimental+Leishmania+donovani-induced+hepatic+granulomas+detects+Kupffer+cell-restricted+antigen+presentation+to+antigen-specific+CD8+T+cells.">Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.</a> PLoS Pathog. 6 (3), e1000805 (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 (6002), 362-366 (2010).</li><li>Vanheule, E., 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=An+intravital+microscopic+study+of+the+hepatic+microcirculation+in+cirrhotic+mice+models:+relationship+between+fibrosis+and+angiogenesis.">An intravital microscopic study of the hepatic microcirculation in cirrhotic mice models: relationship between fibrosis and angiogenesis.</a> Int J Exp Pathol. 89 (6), 419-432 (2008).</li><li>Jenne, C. N., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+surveillance+by+the+liver.">Immune surveillance by the liver.</a> Nat Immunol. 14 (10), 996-1006 (2013).</li><li>Zimmermann, H. W., Tacke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+chemokine+pathways+and+immune+cell+infiltration+as+a+novel+therapeutic+approach+in+liver+inflammation+and+fibrosis.">Modification of chemokine pathways and immune cell infiltration as a novel therapeutic approach in liver inflammation and fibrosis.</a> Inflamm Allergy Drug Targets. 10 (6), 509-536 (2011).</li><li>Kim, J. 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=Two-photon+laser+scanning+microscopy+imaging+of+intact+spinal+cord+and+cerebral+cortex+reveals+requirement+for+CXCR6+and+neuroinflammation+in+immune+cell+infiltration+of+cortical+injury+sites.">Two-photon laser scanning microscopy imaging of intact spinal cord and cerebral cortex reveals requirement for CXCR6 and neuroinflammation in immune cell infiltration of cortical injury sites.</a> J Immunol Methods. 352 (1-2), 89-100 (2010).</li><li>Karlmark, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+recruitment+of+the+inflammatory+Gr1++monocyte+subset+upon+liver+injury+promotes+hepatic+fibrosis.">Hepatic recruitment of the inflammatory Gr1+ monocyte subset upon liver injury promotes hepatic fibrosis.</a> Hepatology. 50 (1), 261-274 (2009).</li><li>Heymann, 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=Hepatic+macrophage+migration+and+differentiation+critical+for+liver+fibrosis+is+mediated+by+the+chemokine+receptor+C-C+motif+chemokine+receptor+8+in+mice.">Hepatic macrophage migration and differentiation critical for liver fibrosis is mediated by the chemokine receptor C-C motif chemokine receptor 8 in mice.</a> Hepatology. 55 (3), 898-909 (2012).</li><li>Ramachandran, P., 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=Differential+Ly-6C+expression+identifies+the+recruited+macrophage+phenotype,+which+orchestrates+the+regression+of+murine+liver+fibrosis.">Differential Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis.</a> Proc Natl Acad Sci U S A. 109 (46), E3186-E3195 (2012).</li><li>Moles, 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=A+TLR2/S100A9/CXCL-2+signaling+network+is+necessary+for+neutrophil+recruitment+in+acute+and+chronic+liver+injury+in+the+mouse.">A TLR2/S100A9/CXCL-2 signaling network is necessary for neutrophil recruitment in acute and chronic liver injury in the mouse.</a> J Hepatol. 60 (4), 782-791 (2014).</li><li>Hammerich, 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=Chemokine+receptor+CCR6-dependent+accumulation+of+&#947;&#948;+T+cells+in+injured+liver+restricts+hepatic+inflammation+and+fibrosis.">Chemokine receptor CCR6-dependent accumulation of &#947;&#948; T cells in injured liver restricts hepatic inflammation and fibrosis.</a> Hepatology. 59 (2), 630-642 (2014).</li><li>Syn, W. -. K., 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=NKT-associated+hedgehog+and+osteopontin+drive+fibrogenesis+in+non-alcoholic+fatty+liver+disease.">NKT-associated hedgehog and osteopontin drive fibrogenesis in non-alcoholic fatty liver disease.</a> Gut. 61 (9), 1323-1329 (2012).</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=Interaction+of+CD44+and+hyaluronan+is+the+dominant+mechanism+for+neutrophil+sequestration+in+inflamed+liver+sinusoids.">Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids.</a> J Exp Med. 205 (4), 915-927 (2008).</li><li>Egen, J. 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The authors disclose no con&#64258;ict of interests.

我々の研究の目的は、肝臓の生体TPLSMイメージングのための高度に標準化された安定した再現性のある方法を開発することであった。一般的に、生体内イメージングは​​、開発、恒常性および疾患における異なる白血球集団のホーミングとの相互作用を、以下の実際の生活条件の下で細胞挙動に貴重な洞察を与えている。しかし、呼吸器および腸の蠕動運動を直接他の固形臓器と比較して?...

細胞や器官全体での細胞機能、さらには生物全体の可視化は、本体2の事実上すべての部分を含め、50年以上のために大きな関心となっている。そのため、いくつかの初期の研究では、すでに肝臓3,4の生体内イメージングを採用。しかし、いくつかの制限が肝組織の長期安定した高解像度画像に関する最新に存在する。 によるダイアフラムおよび胃腸管5との密着肝臓の解剖学的位置に、微細な生体イメージングのための最も一般的な問題は、呼吸による動きと、より少ない程度に、腸管の蠕動6である。他の固形臓器と比較して、肝臓手術は特に困難である。による緻密な微小血管構造のために、外科的処置は、大規模な出血性病変を引き起こす障害微小7、また居住者のiの活性化できそのようなクッパー細胞8としてmmune細胞。したがって、組織の機械的固定は6,9は生体顕微鏡イメージングに干渉する可能性がある他の場所で公開されている。 健康な肝臓では、全血液量の10〜15％は、肝血管系内に存在し、臓器循環の変化（ 例えば 、血圧変動の影響を非常に受けやすい臓器をレンダリングする、全体的な心拍出量10の約25％を受け取る）。したがって、過度の組織処理または集中循環により、例えば 、せん断応力、変位、損傷に対する肝臓の血流の途絶は、同様に肝臓の免疫応答に影響を与え、白血球遊走行動における人為的な変更、障害、肝酸素したがってさらに肝臓の損傷につながる臓器保存および動物の全体的な生活時間など。 初期の微視的研究は、生体内エピ蛍光MIに基づいていたcroscopy、そのような光退色および低い浸透深さのようないくつかの技術的な制約は、長期の肝臓イメージング4,11,12のためのこの技術の使用を制限する。この新しい方法は、現実の状況13-15の下に、ほぼすべての臓器においてイメージング研究を実行することが技術的に可能であったように、1990年代の多光子顕微鏡の発展に伴い、フォト漂白または侵入深さの制限は主に、解決された。しかし、肝臓イメージングに対する主残された課題であった：いくつかの時間の16のより長い期間の呼吸運動、肝臓組織の自己蛍光、肝類洞に不変の血流を確保し、特に安定したイメージング。 いくつかの研究は、肝臓17における様々な白血球の機能および移動に対処し、 例えば 、NKT細胞18-20、T細胞21,22、肝臓マクロファージ23,24または好中球25、長期多メートルもののicroscopyイメージングはまだ成功し、さらに難しい既存の損傷に起因する急性または慢性の肝疾患を有する動物におけるタスク及び更なる損傷26に、したがってより高い感受性を確立されていなかった。しかし、リアルタイムで回遊行動と肝臓の白血球の細胞機能を監視することは、肝臓の恒常性と疾患27において、その特定の役割で小説洞察を可能にする。 ケモカイン受容体CXCR6は、ナチュラルキラー（NK）細胞、NKT細胞、およびいくつかのT細胞集団18,28を含むいくつかのリンパ球サブセット上で発現される。マウスでの以前の研究は、CXCR6とその同族リガンドCXCL16が恒常性の間に肝正弦波にNKT細胞のパトロールを制御することができることが示されている。したがって、（CXCR6座に緑色蛍光タンパク質[gfpを]のノックインを運ぶ）CXCR6.gfpマウスの使用は、脳29のような種々の器官でのリンパ球の遊走を調べるために記載されているまた、肝臓18,20は 、炎症時にCXCR6.gfp細胞の浸潤を増加を示す。 この研究で提供される方法では、安定化条件下で長期間にわたりこれらのプロセスに従うことが可能であった。生体多光子ベースの手順は、動物や器官の最小限の摂動と高度に再現性があったイメージングを可能にした。呼吸と循環の開閉制御に続く大規模な監視により、長期的な動物の生存のために最適化。と非常に柔軟で、腎臓や脾臓などの他の実質臓器にも採用しやすい。

<table border="0" cellpadding="0" cellspacing="0" width="1184">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Anesthetics</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td style="width:293px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Buprenorphine</td>
			<td style="width:263px;">Essex Pharma</td>
			<td style="width:219px;">997.00.00</td>
			<td>Analgeticum, 0.1 mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fentanyl</td>
			<td style="width:263px;">Rotex Medica</td>
			<td style="width:219px;">charge: 30819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fluovac anesthesia system</td>
			<td style="width:263px;">Harvard Apparatus</td>
			<td style="width:219px;">34-1030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Glucose 5%</td>
			<td style="width:263px;">Braun</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ISOFLO (Isoflurane Vapor) vaporiser</td>
			<td style="width:263px;">Eickemeyer</td>
			<td style="width:219px;">4802885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Isoflurane</td>
			<td style="width:263px;">Forene Abbott</td>
			<td style="width:219px;">B 506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Isotonic (0.9%) NaCl solution</td>
			<td style="width:263px;">DeltaSelect GmbH</td>
			<td style="width:219px;">PZN 00765145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Ketamin 10%</td>
			<td style="width:263px;">ceva</td>
			<td style="width:219px;">Charge: 36217/09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Xylazin 2%</td>
			<td style="width:263px;">medistar</td>
			<td style="width:219px;">Charge: 04-03-9338/23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;"></td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Consumable supplies</td>
			<td style="width:263px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">20ml Syringe</td>
			<td style="width:263px;">BD Plastipak</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250ml Erlenmeyer flask</td>
			<td style="width:263px;">Schott Duran</td>
			<td>21 226 36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">25mL Beaker 2x</td>
			<td style="width:263px;">Schott Duran</td>
			<td style="width:219px;">50-1150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">2ml syringe</td>
			<td style="width:263px;">BD Plastipak</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-0 Vicryl suture</td>
			<td style="width:263px;">Ethicon</td>
			<td style="width:219px;">V7980</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Agarose</td>
			<td style="width:263px;">commercially available</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Bepanthen Eye and Nose ointment</td>
			<td style="width:263px;">Bayer Vital GmbH</td>
			<td style="width:219px;">6029009.00.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Change-A-Tip Deluxe High-Temp Cautery Kit</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">18010-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cotton Gauze swabs</td>
			<td style="width:263px;">Fuhrmann GmbH</td>
			<td style="width:219px;">32014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cover Slip 24x50mm</td>
			<td style="width:263px;">ROTH</td>
			<td style="width:219px;">1871</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Durapore silk tape</td>
			<td style="width:263px;">3M</td>
			<td style="width:219px;">1538-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Feather disposable scalpel</td>
			<td style="width:263px;">Feather</td>
			<td style="width:219px;">02.001.30.011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Fine Bore Polythene Tubing 0,58mm ID</td>
			<td style="width:263px;">Smiths medical</td>
			<td style="width:219px;">800/100/200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Histoacryl</td>
			<td style="width:263px;">Braun</td>
			<td style="width:219px;">1050052</td>
			<td>5x 0,5ml</td>
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			<td height="21" style="height:21px;width:411px;">Poly-Alcohol Haut&#8230;farblos Antisepticum</td>
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			<td style="width:219px;">72PAH200</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sterican needle 18 G x 1</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;">304622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sterican needle 27 3/4 G x 1</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;">4657705</td>
			<td></td>
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			<td></td>
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			<td height="21" style="height:21px;">Dumont No.7 forceps</td>
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			<td height="21" style="height:21px;width:411px;">Graefe forceps curved x1</td>
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			<td style="width:219px;">11050-10</td>
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			<td height="21" style="height:21px;width:411px;">Semken forceps</td>
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			<td height="21" style="height:21px;width:411px;">Small surgical scissors curved</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Small surgical scissors straight</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">14028-10</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Standard pattern forceps</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
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			<td height="21" style="height:21px;">Vannas spring scissors</td>
			<td style="width:263px;">Fine Science Tools Inc.</td>
			<td style="width:219px;">15000-08</td>
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			<td height="21" style="height:21px;">Equipment</td>
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			<td height="21" style="height:21px;width:411px;">ECG Trigger Unit</td>
			<td style="width:263px;">Rapid Biomedical</td>
			<td style="width:219px;">3000003686</td>
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			<td height="21" style="height:21px;width:411px;">MICROCAPSTAR End-Tidal Carbon Dioxide Analyzer</td>
			<td style="width:263px;">AD Instruments</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Minivent Typ 845</td>
			<td style="width:263px;">Harvard Apparatus</td>
			<td>73-0043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Multiphoton microscope Trimscope I</td>
			<td style="width:263px;">LaVision</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Perfusor Compact</td>
			<td style="width:263px;">B. Braun</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">PowerLab 8/30 8 channel recorder</td>
			<td style="width:263px;">AD Instruments</td>
			<td style="width:219px;">PL3508</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Temperature controlled heating pad</td>
			<td style="width:263px;">Sygonix</td>
			<td style="width:219px;">26857617</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Temperature sensor</td>
			<td style="width:263px;">comercially available</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Temperature controlled System for Microscopes -Cube&#38;Box</td>
			<td style="width:263px;">Life Imaging Services</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using cxcr6.gfp reporter mice

<html>傷害に応答して肝臓の炎症は、単球、好中球、T細胞サブセット、B細胞、ナチュラルキラー（NK）およびNKT細胞を含む白血球の異なるサブタイプの浸潤を伴う非常に動的なプロセスである。免疫細胞の遊走を監視するための肝臓の生体内顕微鏡起因試料調製及び固定、光学分割および長期動物の生存に関して高い要件に特に困難である。しかし、炎症過程の動力学ならびに細胞の相互作用の研究は、より良好な炎症性肝疾患の開始、進行および退行を理解するために重要な情報を提供することができる。したがって、高感度で信頼性の高い方法は、集中治療と組み合わせて生体二光子レーザー走査顕微鏡（TPLSM）で長時間（約6時間）マウスの肝臓に移行し、異なる免疫細胞の細胞間相互作用を研究するために設立された監視。 ENT &quot;&gt;提供される方法は、穏やかな調製および器官の最小の摂動と肝臓の安定した固定を含み、長期生体内イメージング最大6時間の期間にわたって事実上退色や光毒性効果を多色多光子顕微鏡を用いて、トラッキングを可能にする特定の白血球サブセットの、及び安定した撮影条件によるマウスの重要なパラメータと循環、温度およびガス交換の安定化の大規模な監視する。 肝臓の炎症の際のリンパ球の遊走を調べるためには、ノックインマウスCXCR6.gfpベースライン条件下および四塩化炭素（CCl 4）の腹腔内注射（複数可）によって誘発される急性および慢性肝障害の後に生体内肝臓イメージングに供した。 、ナチュラルキラー（NK） - - このようなCD4 T細胞などのTリンパ球のサブセットでなく、粘膜associ CXCR6、主にナチュラルキラーT（NKT）上で、リンパ球上に発現されるケモカイン受容体であるatedの不変（MAIT）T細胞1。 CXCR6.gfp +免疫細胞の回遊パターンと位置決め後の肝臓損傷の際、変更された行動を詳細に洞察力、したがって、疾患の進行におけるそれらの潜在的な関与を許可。

私たちの生体内TPLSMアプローチを検証するために、我々は生体内TPLSMイメージングにCXCR6のGFP / +マウスを受ける。マウスは、いずれかのベースライン対照として未処理のまま、または急性肝障害20を誘導するために四塩化炭素（CCl 4）の単回腹腔内注射を行った。 ビデオシーケンスは、2-5時間の期間にわたり採取し、細胞は、それらの緑色蛍光を経...

Watch this Scientific Journal Video about Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice at JoVE.com

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

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, ...

long-term-intravital-multiphoton-microscopy-imaging-immune-cells

Research

JoVE Journal

Immunology and Infection

11.8K ビュー.  RWTH University-Hospital Aachen. 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.

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

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

細胞星状に7つのステップ

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

免疫・感染学

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

2光子顕微鏡を用いてマウスの胸腺の生体内イメージング

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

長期的な慢性<em&gt;緑膿菌</emマウスにおける&gt;気道感染

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

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 ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

以下の肝臓の生体顕微鏡イメージング<em&gt;リーシュマニア</em&gt;感染症：肝血行動態の評価

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

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 ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

解析<em&gt;腸炎エルシニア</em&gt;エフェクター転β-ラクタマーゼエフェクター融合を用いて宿主細胞に

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

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

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

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

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

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

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

Intravital Imaging of Neutrophil Priming Using IL-1&amp;#946; Promoter-driven DsRed Reporter Mice

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

IL-1βプロモーター駆動のDsRedレポーターマウスを用いた好中球プライミングの生体内イメージング

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

レポーター酵素蛍光を用いたマウスにおける結核菌をイメージング

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...