著者は、私たちはリンパ節のスライスを実行するよう奨励博士アラントラウトマンに感謝したいと思います。この作品は、リーグ国立Contreルがん、財団原子核Médicaleのフランスと連合原子核シュルルのがんからの補助金によって部分的にサポートされていました。

1。リンパ節からのスライスの準備<ol><li>埋め込みには4％低融点アガロース溶液を調製。 PBSおよびマイクロ波このソリューションでアガロースを希釈する。アガロースが完全に溶解したら、° C準備が整うまでの使用のために37のソリューションを維持する。 </li><li> 6ウェル組織培養プレートを準備します。ウェルあたり1.1ミリリットルRPMI完全培地を追加し、各ウェルにおける器官型フィルタを挿入する。 4℃で6 - ウェルプレートを置き、スライスを転送する準備までのC。 </li><li> RPMI完全培地で満たされたプラスチック製の皿に、4 mmの内径で、ステンレス製のワッシャーを置きます。ワッシャーはさらにビブラトームカットのスライス上に細胞を濃縮するために使用されます。 </li><li>地元の動物福祉の規則に従って、マウスを犠牲にする。慎重に周囲の組織から末梢リンパ節を切除すると、氷冷したPBSを含むプラスチック製の皿に置いてください。これらの器官は非常にソフトなので注意は、リンパ節の構造に損傷を与えないように注意する必要があります。 </li><li> 35mmプラスチックシャーレに4％アガロースゲルを注ぐと繊細なゲルにノードを移す。硬化まで5分間氷上でアガロースゲルを残す。 </li><li>皿からブロックを削除し、各リンパ節の周囲にゲルの3〜5 mmを残すために寒天をトリミング。 </li><li>無毒の組織接着剤でビブラトームの試料のディスク上に埋め込まれたノードを取り付けます。氷冷PBSで満たされたトレイに試料ディスクを取り付けます。 </li><li>セクション遅い範囲（0.3 mm / sの）と中距離（1.5 mm）に設定された振動の周波数に設定さビブラトーム速度320μmの厚さで寒天包埋組織。 </li><li>それらは微細な鉗子を用いて器官培養用インサートの上にカットされているように慎重にリンパ節のスライスを転送する。各インサートの3スライスを置きます。スライスが簡単に破損することができるように細心の注意を使用してください。 320μmでカットするときに典型的な末梢リンパ節は、5つのセクションが得られます。彼らは表面的な組織を含んでいるので、最初と最後のスライスを捨てる。 </li><li>個々のスライスにステンレス製のワッシャーを置きます。ワッシャーはよく組織を取り巻くアガロース上に配置されていることを確認します。 </li><li> 37℃で培養プレートをインキュベート℃、5％CO 2の加湿インキュベーター中でCプレートT細胞への準備ができるまで。 </li></ol> 2。マウスのリンパ節からT細胞を単離する<ol><li>以前に発行されたJoveの第3条によれば、T細胞を分離する。 </li><li>すぐにT細胞を使用して、次のセクションに進んでください。そうでなければ、リンパ球は10 ng / mlのIL - 7を添加したRPMI -培地を完全に一晩培養される可能性があります。 </li></ol> 3。分離されたT細胞のラベリング<ol><li> HBSSで細胞を洗浄。 ml当たり10 × 10 6で同じバッファーで再懸濁します。 </li><li> 0.5μMの最終濃度を与える1μMセルトラッカーグリーンCMFDAを含むHBSSの同じボリュームを追加します。 </li><li> ℃で5分間の間に37℃で細胞懸濁液をインキュベートする。 RPMI -培地を完全に細胞を2回洗浄する。 </li><li> RPMI -培地を完全にmlあたり10 × 10 6の最終濃度で再懸濁し、細胞。これらの標識された細胞は、スライス上にプレーティングされます。 </li></ol> CMFDA以外の蛍光色素は余りに使用されますが、これらの分子は一定の濃度を上記の潜在的に有毒であることに留意してください可能性がある。 4。メッキは、リンパ節のスライスの上にT細胞を標識し<ol><li>ピペットとワッシャーの内側の一部に含まれる余分な培地を削除します。組織が乾燥しないようにしてください。このステップですぐに働く。 </li><li>ゆっくりとワッシャの内側の部分に1〜2 × 10 5細胞に相当する10〜20μlのラベルされたT細胞を分注する。ケアは、ピペットの先端で組織に触れないように注意する必要があります。細胞懸濁液の滴が所定の位置に留まることを確認してください。 </li><li> 37℃インキュベーターで細胞とスライスを配置℃、5％リンパ球が組織に移行できるように、少なくとも30分間CO 2。 </li></ol> 5。リンパ節のスライス内イメージングT細胞このビデオの記事で、我々広視野倒立顕微鏡と共焦点正立顕微鏡でリンパ節スライスの画像蛍光T細胞。 <ol><li> 37℃の温度を℃に設定私たちの顕微鏡は、温度制御されたチャンバーを備えています。 </li><li>酸素（5％CO 2、95％O 2）フェノールレッドを含まないRPMI培地でリンパ節のスライスの連続的な灌流を可能にする灌流システムをインストールします。私たちのシステムでは、潅流媒体は重力によって片側から撮影室に入ります。培地は、廃棄物の収集フラスコに接続されたポンプで吸引される。メディアの流速1ml /分に設定します。 </li><li>細かい鉗子で、微妙に6 - ウェルプレートからスライスを取り出し、組織に浸透しない蛍光細胞を取り除くために数秒間温め、酸素RPMI培地中で準備をディップ。 </li></ol> IMA広視野倒立顕微鏡でT細胞パトンの<ol start="4"><li>ガラスボトムディッシュの内側の一部に接着されたナイロン糸で構成されてカスタムメイドのイメージングチャンバーに逆さにスライスを置きます。このチャンバーでは、ナイロン糸、ガラスからスライスを昇格し、組織に拡散する酸素ソリューションを有効にします。リンパ節におけるT細胞の遊走は、酸素に強く依存するとして、これは必須です。その上にステンレス製のワッシャーを追加してスライスを固定します。これらの条件では、スライスには、灌流液の更新を容易に皿の底部上に数ミクロンの場所にあります。 </li><li>広い視野（800 × 800μmの、ノードのスライス面の約3分の1）をキャプチャするには、10倍、乾燥対物レンズを使用して画像を収集する。我々は10倍ニコンSフッ素0.5 NAは我々の分析に最も適していたことがわかった。 </li></ol>典型的なタイムラプスイメージングの実験は、軸方向（z）の寸法が50μmの合計深さにまたがる5オプティカル平面をキャプチャします。蛍光T細胞は、通常10〜20分中10〜30秒までの間隔で撮像される。 共焦点正立顕微鏡とイメージングT細胞このプロトコルで使用されている共焦点顕微鏡は20倍の水浸対物レンズ（オリンパス、20x/0.95 NA）を搭載したライカSP5です。 <ol start="6"><li>顕微鏡のイメージング段階にスライスを確保する。注：私達は通常それを固定するために準備にワッシャーを配置。 </li><li> z軸に沿って一連の画像を収集することにより、イメージングセッションを設定します。無傷の組織構造内の画像のT細胞に、開始位置は、通常、最初のラベルの付いたT細胞より5〜10μm程度に設定されています。 </li></ol> 6。代表的な結果このビデオプロトコルに従うことで、ノードのTゾーン、通常は生体内でこの特定の環境（図1）で起こる現象に蓄積された蛍光性T細胞の多数を可視化することを期待してください。特に、T細胞は、ちょうどカプセルの下に通常あるB細胞のゾーン、から除外する必要があります。成功した実験はまた、無傷のリンパ節4で得られた公表された結果と整合性の高い運動性の行動（図3）を表示し、組織（図2）に勧誘T細胞になります。平均では、スライス内の個々のT細胞の平均速度は近い〜10μm/分（図4）でなければなりません。 <img src="/files/ftp_upload/3054/3054fig1.jpg" alt="図1"/> 図1。 T細胞はリンパ節のスライスに蓄積する。蛍光標識T細胞（CMFDA、緑）画像取得の前にリンパ節のスライス30分（上写真）に追加された。画像は、z方向に50μmをまたがる5枚の画像の最大投影です。明視野像は、底部に示されています。画像は広視野顕微鏡で取得されたものです。 <img src="/files/ftp_upload/3054/3054fig2.jpg" alt="図2"/> 図2。 T細胞はリンパ節のスライスに採用されています。蛍光標識T細胞（CMFDA、緑）共焦点顕微鏡を用いて画像取得の前にリンパ節のスライス30分に追加された。画像は切断面（上写真）と、以下の40μmの（下の写真）で捕獲された。 <img src="/files/ftp_upload/3054/3054fig3.jpg" alt="図3"/> 図3。 T細胞はリンパ節のスライス内で非常に運動性があります。蛍光標識T細胞は広視野顕微鏡を使用して12分間で画像化した。個々のT細胞の軌跡は、（高運動性細胞）赤青（低運動性の細胞）から増加する変位を表現するために色分けされたトラックとして表示されます。トラックはImarisソフトウェアを用いて計算した。破線の楕円は、推定されるB細胞のゾーンを区切るのに対し、白い線は、ノードのエッジに従います。 <img src="/files/ftp_upload/3054/3054fig4.jpg" alt="図4"/> 図4。リンパ節のスライス内では、T細胞の速度は10μm/分の近くにあります。速度を図3に表されたトラックからImarisソフトウェアを用いて計算した。

<ol>
	<li>Asperti-Boursin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCR7+ligands+control+basal+T+cell+motility+within+lymph+node+slices+in+a+phosphoinositide+3-kinase-independent+manner.">CCR7 ligands control basal T cell motility within lymph node slices in a phosphoinositide 3-kinase-independent manner.</a> J Exp Med. 204, 1167-1167 (2007).</li><li>Bhakta, N. R., Oh, D. Y., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+oscillations+regulate+thymocyte+motility+during+positive+selection+in+the+three-dimensional+thymic+environment.">Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment.</a> Nat Immunol. 6, 143-143 (2005).</li><li>Matheu, M. P., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CD4++T+cells+from+mouse+lymph+nodes+using+Miltenyi+MACS+purification.">Isolation of CD4+ T cells from mouse lymph nodes using Miltenyi MACS purification.</a> J Vis Exp. , (2007).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <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+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1869 (2002).</li><li>Ehrlich, L. I., Oh, D. Y., Weissman, I. L., Lewis, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contribution+of+chemotaxis+and+substrate+restriction+to+segregation+of+immature+and+mature+thymocytes.">Differential contribution of chemotaxis and substrate restriction to segregation of immature and mature thymocytes.</a> Immunity. 31, 986-986 (2009).</li><li>Gollmer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCL21+mediates+CD4++T-cell+costimulation+via+a+DOCK2/Rac-dependent+pathway.">CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway.</a> Blood. 114, 580-580 (2009).</li></ol>

利害の衝突は宣言されません。

我々は、導入T細胞の挙動を調査するために使用されるリンパ節のスライスを、生成するための簡単​​な、迅速かつ堅牢なテクニックを説明してきました。近年では、このメソッドは、T細胞の位置と運動性1,5を制御する細胞外因子を同定するために胸腺およびリンパ節のスライスを使用して正常に適用されています。また、正の選択時および抗原認識2,6時にT細胞の胸腺細胞でのCa 2 +応答を測定するために使用されています。オーバーレイのスライスのアッセイは、議論されるに値する利点と制限があります。必要なものはは、細胞遊走の分子制御を妨害するために、薬理学的にスライスを操作する場合注目すべきは、このシステムでは有用な、組織へのアクセスを許可します。画像に続いて、スライスが撮像されている構造についてのさらなる情報を収集するために、免疫組織化学のために処理することができます。また、観察は、従来の広視野の蛍光顕微鏡で行うことができます。解像度は、共焦点または二光子顕微鏡とほど良好ではないとその光毒性は原則的に二光子顕微鏡による複数の光子とのより深刻な場合、それは単純さ、低コスト、そしてより大きな選択の利点がありますが、励起波長の。 無傷のリンパ節におけるT細胞のイメージングは​​、そのリンパ器官に続いてホームラベル付けリンパ球の静脈注射を必要とします。我々は以前に1を示しているように、スライスのアッセイは、養子移入実験と完全に互換性があります。しかし、リンパ節へホームへのT細胞に必要な時間の長さは、時間の経過とともに細胞外に漏出する傾向を持っている蛍光染料の使用を複雑にすることができます。これは、Ca 2 +染料フラ-2のケースです。組織へのT細胞の迅速な採用（&lt;30分）で、スライスのアッセイは、このような染料を使用する機会を提供します。最後に、この方法はいくつかのヒト組織でのT細胞機能を解析することには大きな利点が生きて維持している。 この実験系では、留意する必要があるにも制限があります。スライスに関連付けられている被害は、特に切断面付近の組織の表面的な領域で、T細胞の機能に影響を与える可能性があります。スライス内の細胞死を評価するために、我々は、標的の原形質膜と細胞を貫通する蛍光染料SYTOXの緑を使用している。我々の実験は、主に組織の表層領域に局在総リンパ節細胞、、の約20％が蛍光この核の色素で標識されたことが明らかになった。アッセイのもう一つの潜在的な懸念は、ケモカインなどの重要な可溶性因子を保持するスライスの能力です。 、我々はT細胞がスライス内で良好な運動性を表示するので、私たちのデータは、このような問題の影響を受けている兆候がないが、我々は、切断面から数十μmにある健康な地域でのイメージングT細胞の重要性を強調したい。これはこのプロトコルのような伝統的な顕微鏡（広視野または共焦点）で行うことができるのに対し、それはリンパ節のスライス標本は、2光子イメージングは​​深さで空間分解能を向上させ、光毒性を低減すると結合される可能性があります。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td>共焦点顕微鏡</td><td>ライカ</td><td> SP5 </td><td></td></tr><tr><td>ビブラトーム</td><td>ライカ</td><td> VT1200S </td><td></td></tr><tr><td>ファイン鉗子</td><td>世界の精密機器</td><td> 14142 </td><td></td></tr><tr><td> 30mmの文化の挿入</td><td>ミリポア</td><td> PICM0RG50 </td><td></td></tr><tr><td> RPMI </td><td>インビトロジェン</td><td> 61870010 </td><td>完全RPMI -培地は10％熱不活性化ウシ胎児血清およびペニシリン/ストレプトマイシンを添加することにより行われる</td></tr><tr><td>ハンクス平衡塩類溶液（HBSS） </td><td>インビトロジェン</td><td> 14170088 </td><td></td></tr><tr><td>低ゲル化温度アガロース、タイプVII - </td><td>シグマアルドリッチ</td><td> A0701 </td><td></td></tr><tr><td>ブチルシアノアクリレート接着剤、Vetbond </td><td> 3M </td><td> 1469 </td><td></td></tr><tr><td>ステンレススチールワッシャー、内径4mmの</td><td> DIYストア</td><td></td><td></td></tr><tr><td>セルトラッカーグリーンCMFDA </td><td>インビトロジェン</td><td> C7025 </td><td></td></tr></tbody></table>

ex vivo imaging of t cells in murine lymph node slices with widefield and confocal microscopes

まれに発現する抗原を検出するために末梢リンパ節、を含む、二次リンパ器官へのナイーブT細胞が継続的にトラフィック。リンパ節にT細胞の遊走はケモカインを含む細胞と化学の両方の要素を含む複雑なプロセスです。最近、二光子顕微鏡の使用は、無傷のリンパ節にT細胞を追跡するために、他の細胞と彼らの行動とその相互作用にいくつかの定量的な情報を得るために許可しています。 in vivo系への明らかな利点がありますが、このアプローチは、複雑で高価な計測機器を必要とし、組織への制限されたアクセスを提供します。マウスリンパ節内T細胞の挙動を分析するために、我々は、スライスのアッセイ1、もともとneurobiologistsが立ち上げてマウス胸腺2に、最近転置を開発しました。この手法では、蛍光標識T細胞は、急性準備リンパ節のスライスの上にメッキされています。このビデオの記事では、組織へのT細胞の局在と移行は、広視野と共焦点顕微鏡でリアルタイムに分析されます。 in vivoで二光子顕微鏡は、自然環境の中で画像のT細胞への効果的なアプローチを提供し、T細胞の移動のメカニズムを解明するために補完する技術。

Watch this Scientific Journal Video about Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes at JoVE.com

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

このプロトコルは、リンパ節のスライスに導入された画像蛍光T細胞への方法を説明します。技術は、従来の広視野蛍光または共焦点顕微鏡でのT細胞遊走のリアルタイム分析を可能にします。

広視野と共焦点顕微鏡によるマウスリンパ節スライスにおけるT細胞のex vivoでのイメージング

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

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Immunology and Infection

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

16.7K ビュー.  Université Paris Descartes, CNRS (UMR 8104). このプロトコルは、リンパ節のスライスに導入された画像蛍光T細胞への方法を説明します。技術は、従来の広視野蛍光または共焦点顕微鏡でのT細胞遊走のリアルタイム分析を可能にします。

ビデオ: Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

マウスT細胞におけるT細胞受容体のレトロウイルス導入

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

免疫・感染学

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

鼠径リンパ節の生体顕微鏡

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

マウスの表在性リンパ節手術

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

マウス膝窩リンパ節の生体内イメージング

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1感染CD4の生得センシングを評価<sup&gt; +</sup&gt;使用して形質細胞様樹状細胞によるT細胞<em&gt;エクスビボ</em&gt;共培養系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

ネズミリンパ節間質細胞の単離

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

アン<em&gt;エクスビボ</em急性脳スライスにおけるオリゴデンドロサイト指向T細胞攻撃の&gt;モデル

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

単純ヘルペスウイルス1型を持つマウス表皮の ex vivo 感染

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.