私たちは、プロトコルの重要な読書のためのシルヴィージャンルサージとすべてのラボのメンバーに感謝します。この作品は、健康の研究（CIHR）のカナダの協会からの助成金（MOP-77545）によってサポートされていました。ナタリーLabrecqueはFRSQシニア奨学金によってサポートされており、メリッサマチューはNSERCアレクサンダー·グラハム·ベル·カナダ大学院奨学金を受けています。

1。手術前の準備マウスは、アニマルケアのガイドラインのカナダ人評議会に従って処理した。 <ol><li>ケタミン/キシラジン（150/300 mg / kg投与、ip）を注入するか、またはイソフルランの吸入（2％、1Lの酸素）によるAnesthetiseマウス。可能であればそれは麻酔時間と深さのよりよい制御を可能にするとして、イソフルランを使用する必要があります。また、酸素は動物の生理機能をサポートし、同時に投与される。また、イソフルランは、直接肺によって除去されるため、肝臓または腎臓の代謝を必要としません。 </li><li>または他の鎮痛剤 - ブプレノルフィン（0.1 mg / kg体重、皮下0.05）注入します。 </li><li> （イソフルランが使用されている場合）眼の乾燥を避けるために手術前にマウスの目に軟膏を適用します。 </li><li>マウスは、その足をつまんで、そのしっぽを丸めて眠っていることを確認します。マウスが反応しない場合は、手術を続行します。 </li></ol> 2。外科<pクラス= &quot;jove_content&quot;&gt;これは、手術による表在リンパ節（LN）の4を削除することが可能である：2鼠径部と2上腕。古いマウスの脂肪組織の存在は、LNの分離を妨げることができるのでLNの単離は、若いリーンマウス（6-8週）の方が簡単です。しかし、この制限は、反復練習で克服されています。マウスの快適さのために、我々は時間で2 LNSの最大の収穫、我々はタイムコース実験を行うとき、我々は一日おきにLNSを収穫。また、感染を防ぐために、抗生物質をマウスに与えられたが、我々はしないと傷がうまく治癒されている可能性があります。一つは、実験デザインに応じて、抗生物質の使用は（細菌への応答が測定される場合など）が観察結果に影響を与える可能性があることに注意する必要があります。すべての楽器がそれぞれの使用後に滅菌され、無菌状態は、手続きを通して維持されることに注意してください。 <ol><li>その側にマウスを置きます。 </li><li>あなたがincisioを実行する領域をローカライズするnは、あなたが収穫したいLNによって異なります。 （ 図1）。 </li><li>その領域に、クロルヘキシジン、または他の消毒剤を適用します。 </li><li>前足の裏に、腹部の近くに後足（ 図1）上記のいずれかを鋭いハサミで小さな切開（約5mm）を作る。 </li><li>あなたは鉗子で切断した緩い毛を削除します。必要であれば、切開を行う前に、マウスを剃ることができます。しかし、それは手順が長く、我々はここに示すように、それは両方とも非常に稀である治癒または減少感染症を改善しないので、それは必要がないことを見つける。 </li><li> LNを見るために2ピンセットで切開を伸ばす。切開は10 mmに達することがあります。 LNは灰色がかったまたは周囲の脂肪よりも暗く表示されます。 </li><li> 1鉗子とLNの上に筋膜（脂肪と組織を網羅薄い膜）をつまんで、周囲の組織を壊さずに軽く引っ張ります。 </li><li>可能な限りunde第二鉗子を配置LNをrneath。第一鉗子を使用すると、筋膜を破るとLNを削除します。あなたが正常にLNを収穫している場合は、血液の汚れは、LNが以前にあった場所に表示されます。 LNは、等張液に沈むことに注意してください。この単純なテストは、あなたがLNではなく、脂肪組織を抽出していることを検証することができます。 </li><li>全く毛が傷口に入っていないことを確認します。 </li><li> （皮膚の内側）を一緒に皮膚の2つの部分を張り付けることで、クリップでそれらをステープルで切開を閉じます。我々は、適用し鉗子でいくつかのミシェル·クリップを使用しています。切開の大きさに応じて、1つまたは2つのクリップを取り付けます。組織が回復するときに最も頻繁に、クリップが脱落します。 </li></ol> 3。術後処置<ol><li>マウスは、手術後に容易に供給することができるようにケージの底にいくつかのウェットフードを追加します。 </li><li>手術後6から8時間ほど、彼らは痛みを経験していないことを確認するためにマウスを確認してください。慎重に行動（姿勢、歩行、内を観察する毛皮の同族と外観teraction）。マウスの全体的な外観が悪い場合には、ブプレノルフィンの2回目を管理します。次の日でマウスは、ブプレノルフィンの投与を継続するそうでなければ彼らの正常な動作を取り戻す必要があります。マウスは以前動物·ケア協議会によって決定され、受け入れられるエンドポイントに到達することはありません。あなたの動物のケアのビバリウムの技術者からアドバイスや助けを求めることをお勧めします。 </li></ol> 4。リンパ節の取り扱い<ol><li>研究室では、単一の細胞懸濁液を得るために、通常の手順に従って、LNを解離させる。我々は、すりガラスのスライドに対して、LNを押すと、定期的にLNあたり1-2×10 6細胞を得る。また、我々は、RNA抽出のためにLNを準備します。 </li><li>あなたの通常のプロトコルに従って細胞を染色。あなたが期待LNフローサイトメトリー·プロファイルを取得する必要があります。 </li></ol> 5。代表的な結果前方と側方散乱pの例多くの手術によって、または犠牲マウス（ 図2）から麻酔マウスから採取したLNSのための示されています。プロフィールやライブリンパ球ゲート内の細胞の割合はLN手術は収穫細胞への適切な手法であることを示していると同じです。 <img alt="図1" src="/files/ftp_upload/3444/3444fig1.jpg" /> 図1マウスの鼠径部および上腕LNの局在。 A.鼠径LNは少し腹に向かって、ちょうど後足の上に位置しています。 B.上腕LNは、前足の後ろに位置している。 <img alt="図2" src="/files/ftp_upload/3444/3444fig2.jpg" /> リンパ球の図2フローサイトメトリープロファイルは、LNの手術後に得られた。前方と側方散乱プロファイルはanesthから収穫LNSのための示されている手術（）によって犠牲マウス（B）からetizedマウス。 LNSは、解離し、収穫し、フローサイトメーターで分析した。ライブリンパ球ゲート内の細胞のパーセンテージが表示されます。

<ol>
	<li>Zehn, D., Lee, S. Y., Bevan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+but+curtailed+T-cell+response+to+very+low-affinity+antigen.">Complete but curtailed T-cell response to very low-affinity antigen.</a> Nature. 458, 211-214 (2009).</li><li>Vezys, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+CD8+T-cell+compartment+grows+in+size+with+immunological+experience.">Memory CD8 T-cell compartment grows in size with immunological experience.</a> Nature. 457, 196-199 (2009).</li><li>Zhou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+persistence+of+memory+CD8(+)+T+cells+depend+on+T+cell+factor+1.">Differentiation and persistence of memory CD8(+) T cells depend on T cell factor 1.</a> Immunity. 33, 229-240 (2010).</li><li>Wirth, T. C., Badovinac, V. P., Zhao, L., Dailey, M. O., Harty, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+central+memory+CD8+T+cells+is+independent+of+CD62L-mediated+trafficking+to+lymph+nodes.">Differentiation of central memory CD8 T cells is independent of CD62L-mediated trafficking to lymph nodes.</a> J. Immunol. 182, 6195-6206 (2009).</li><li>Rooke, R., Waltzinger, C., Benoist, C., Mathis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+complementation+of+MHC+class+II+deficiency+by+intrathymic+delivery+of+recombinant+adenoviruses.">Targeted complementation of MHC class II deficiency by intrathymic delivery of recombinant adenoviruses.</a> Immunity. 7, 123-134 (1997).</li><li>Witherden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetracycline-controllable+selection+of+CD4(+)+T+cells:+half-life+and+survival+signals+in+the+absence+of+major+histocompatibility+complex+class+II+molecules.">Tetracycline-controllable selection of CD4(+) T cells: half-life and survival signals in the absence of major histocompatibility complex class II molecules.</a> J. Exp. Med. 191, 355-364 (2000).</li><li>Labrecque, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+much+TCR+does+a+T+cell+need.">How much TCR does a T cell need.</a> Immunity. 15, 71-82 (2001).</li><li>Leignadier, J., Labrecque, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitope+density+influences+CD8++memory+T+cell+differentiation.">Epitope density influences CD8+ memory T cell differentiation.</a> PLoS One. 5, e13740 (2010).</li><li>Leignadier, J., Hardy, M. P., Cloutier, M., Rooney, J., Labrecque, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+T-lymphocyte+survival+does+not+require+T-cell+receptor+expression.">Memory T-lymphocyte survival does not require T-cell receptor expression.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 20440-20445 (2008).</li><li>Obst, R., van Santen, H. M., Mathis, D., Benoist, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+persistence+is+required+throughout+the+expansion+phase+of+a+CD4(+)+T+cell+response.">Antigen persistence is required throughout the expansion phase of a CD4(+) T cell response.</a> J. Exp. Med. 201, 1555-1565 (2005).</li><li>Bassett, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T-cell+expansion+and+maintenance+after+recombinant+adenovirus+immunization+rely+upon+cooperation+between+hematopoietic+and+nonhematopoietic+antigen-presenting+cells.">CD8+ T-cell expansion and maintenance after recombinant adenovirus immunization rely upon cooperation between hematopoietic and nonhematopoietic antigen-presenting cells.</a> Blood. 117, 1146-1155 (2011).</li><li>Lacombe, M. H., Hardy, M. P., Rooney, J., Labrecque, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-7+receptor+expression+levels+do+not+identify+CD8++memory+T+lymphocyte+precursors+following+peptide+immunization.">IL-7 receptor expression levels do not identify CD8+ memory T lymphocyte precursors following peptide immunization.</a> J. Immunol. 175, 4400-4407 (2005).</li><li>Leignadier, J., Rooney, J., Daudelin, J. F., Labrecque, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lowering+TCR+expression+on+naive+CD8++T+cells+does+not+affect+memory+T-cell+differentiation.">Lowering TCR expression on naive CD8+ T cells does not affect memory T-cell differentiation.</a> Immunol. Cell. Biol. 89, 322-325 (2011).</li></ol>

利害の衝突が宣言されません。

我々は、多くの実験系に適用することができLN手術のプロトコルを記述している。免疫応答または免疫細胞の恒常性に従うことが非常に有用ですが、このテクニックはいくつかの制約があります。まず、4つのみLNSを収穫することができます。第二に、研究免疫応答は、全身であるか、浅鼠径または上腕（皮排水）LNSで実行する必要があります。最後に、採取した細胞の数は、LNの除去、絶対?...

<table border="1"><tbody><tr><td> 試薬名 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>グレーフェファインパターンプレミアム鉗子</td><td>ハーバード装置</td><td> 52から2144 </td><td>強く湾曲し、10センチ（4インチ）、0.8 mmの先端</td></tr><tr><td>鉗子を適用すると削除ミシェルクリップ</td><td>ハーバード装置</td><td> 52から3779 </td><td> 12.5センチメートル（5） </td></tr><tr><td>ミシェル·クリップ100 </td><td>ハーバード装置</td><td> 52から3746 </td><td> 7.5〜1.75ミリメートル</td></tr><tr><td> Temgesic（ブプレノルフィン塩酸塩） </td><td>シェリング·プラウ</td><td></td><td></td></tr><tr><td> Vetalar（塩酸ケタミン） </td><td> Bonicheアニマルヘルスケア</td><td></td><td> DIN：01989529 </td></tr><tr><td> Rompun（キシラジン） </td><td>バイエル</td> &lt;TD&gt; </td><td> DIN：02169592 </td></tr><tr><td>イソフルレン</td><td> ABBOTT </td><td></td><td> DIN：02032384 </td></tr><tr><td> Hypotearsの眼軟膏</td><td>ノバルティスOphtalmics </td><td></td><td> DIN：02133288 </td></tr><tr><td> Baxedin（イソプロピルアルコールでClorhexidineグルコン酸2パーセント70％） </td><td>オメガ</td><td> L0000017 </td><td> DIN：02251477 </td></tr></tbody></table>

murine superficial lymph node surgery

ワクチン、感染症や腫瘍に対する免疫応答の進行を理解する免疫学の分野では、応答は、しばしば時間をかけて続いています。同様に、リンパ球の恒常性の研究では、時間経過実験をする必要があります。同じマウス内でこれらの研究を実行すると、実験的な変動と同様に使用されるマウスの数を減らすことが理想的です。血液の撤退は時間経過実験のパフォーマンスを可能にする、それが唯一の循環リンパ球に関する情報を提供し、細胞1-4の限られた数を提供しています。体を循環するリンパ節に存在するリンパ球は異なる特性を持っているので、両方の場所を調べることが重要です。手術によるリンパ節の連続除去が免疫応答や時間をかけて同じマウスの免疫細胞の増殖に従うユニークな機会を提供しています。 PEに十分であるリンパ節あたり1×10 6個の細胞の間にさらに、この技術は、収量表現型の特性および/または機能アッセイをrform。シーケンシャルリンパ節の手術やリンパ節郭清が正常に私たちと他の5月11日によって使用されています。ここでは、上腕動脈と鼠径部リンパ節がanesthetisedマウスの皮膚に小切開を行うことにより除去することができる方法について説明します。手術は表面的な、急速に行われるので、マウスは非常に迅速に回復し、よくヒールや過度の痛みを経験していません。一日おきに、それは時間のコースの実験を可能にする1つまたは2つのリンパ節を採取することが可能です。この手法は、時間をかけてリンパ節に存在するリンパ球の特性を研究するため、適しています。このアプローチは、様々な実験デザインに適しており、我々は多くの研究室は、順次リンパ節の手術を行うことの恩恵を受けるだろうと信じています。

Watch this Scientific Journal Video about Murine Superficial Lymph Node Surgery at JoVE.com

Murine Superficial Lymph Node Surgery

同じマウス内で時間をかけて免疫応答の進行を追跡するには、リンパ節は、順次手術によって除去することができる。ここでは、この手法を実行する方法について説明します。

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 ...

Research

JoVE Journal

Immunology and Infection

42.0K ビュー.  Maisonneuve-Rosemont Hospital Research Center. 同じマウス内で時間をかけて免疫応答の進行を追跡するには、リンパ節は、順次手術によって除去することができる。ここでは、この手法を実行する方法について説明します。

ビデオ: Murine Superficial Lymph Node Surgery

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 ...

免疫・感染学

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

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

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

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

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 ...

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 ...

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 ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

弱毒で皮内免疫後のマウスの皮膚及び排出リンパノードから骨髄性細胞の単離<em&gt;マラリア原虫</em&gt;スポロゾイト

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

マウス全層皮膚移植

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

This article provides detailed methodology to identify and quantify functional T lymphocyte subsets present within murine kidney, aorta and lymph nodes by intracellular staining and flow cytometry. The model of angiotensin II induced hypertension was chosen to explain, step-by-step, the procedures and fundamental principles of flow cytometry and intracellular staining.

細胞内染色およびフローサイトメトリーは、高血圧のモデルにおけるマウス大動脈、腎臓及びリンパ節内のリンパ球サブセットを識別するために、

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

コレクションと RNA 解析のための大型動物からリンパ節の処理: リンパ節トランスクリプトーム研究大型動物種のための準備

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

マウス胸腺、膵リンパ節および脾臓フローサイトメトリーを用いた排水における制御性 T 細胞サブセットの定量

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...