The authors thank Sanjiv Luther and colleagues for helpful discussions in establishing the current lymph node digestion protocol. This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R.

このビデオ出版物およびプロトコルでは、すべての動物手順は州立機関バーゼル=シュタット、スイスで承認された動物プロトコルに従って行った。 1。リンパ節の準備と消化<ol><li>加熱プレートとマグネチックスターラー上で37℃にビーカー内の前の熱水。 </li><li>次のような基本的なミディアムを準備したDMEM培地（ピルビン酸なし）、2％FCSを補充し、1.2mMのCaCl 2およびペニシリン/ストレプトマイシン（ペニシリン100単位、ストレプトマイシン100μgの）。 </li><li>使用前にすべての解剖器具を滅菌する。 </li><li> CO 2窒息あたりのリンパ節ドナーマウスを安楽死し、無菌的にリンパ節を解剖。周囲の脂肪を解剖しないでください。注：このプロトコルは、末梢皮膚流入領域リンパ節（鼠径、上腕、腋窩）に最適化されています。 </li><li> 2ミリリットルの氷冷塩基性媒体を含む滅菌ペトリ皿にリンパ節を配置します。 </li><li>リンパ節CAを混乱させる1mlシリンジに固定された2つの25のGの針を使用してpsule。 </li><li> 1 mg / mlのコラゲナーゼIVおよび40μg/ mlのDNアーゼIを補充した750μlの基本培地を含む5ミリリットルのポリプロピレン丸底チューブ内の破壊されたリンパ節組織を転送</li><li>各チューブ内の1つの無菌磁気攪拌機を追加します。 </li><li> 37℃でビーカー内の場所チューブは、水を予熱し、30分間ゆっくりとした速度（1ラウンド/秒）で、チューブをかき混ぜる。 </li><li>加熱板と磁気攪拌機からチューブを外し、リンパ節の断片が安定しましょう​​。 </li><li>慎重に「非間質細胞」が濃縮された上清を除去。注：T、B、樹状細胞およびCD45を分析する場合- GP38 - CD31 -は予見され、「非間質細胞」部分を保存します。 </li><li>洗浄は750μlの基本培地で1回リンパ節組織を残り。注：この手順は、免疫能力のあるマウスを操作する場合にのみ必要です。 </li><li>リンパ節断片が沈降しましょう​​。 </li><li>削除する非間質細胞浮遊割合。 </li><li>リンパ節フラグメントに750μlの基本3.5ミリグラムを添加した培地/ mlのコラゲナーゼDを追加し、40 / mlのDNアーゼI。 </li><li>バックを37℃予熱した水を入れたビーカー内チューブを配置します。 </li><li>ゆっくり攪拌しながら5分間リンパ節組織を消化。 </li><li>ピペッティングによりリンパ節組織断片を分解し、自動マルチチャンネルピペットを用いて最大速度で10サイクル700μlの混合。注：これは消化を改善するためにリンパ節組織を破壊する。 </li><li> 37℃の予熱された水で戻ってビーカー内チューブを配置します。 </li><li>ゆっくりと撹拌しながらさらに10分間、リンパ節組織断片を消化。 </li><li>ピペッティングによりリンパ節組織断片を分解し、自動マルチチャンネルピペットを用いて最大速度で99回混合する。 </li><li>単一細胞懸濁液の維持を確実にするために、0.5MのEDTA 7.5を添加する。 </li><li>パイプによってリンパ節組織断片を分解準自動化されたマルチチャンネルピペットを用いて、最大のスピードで99サイクルで混合。 </li><li> 750μlの塩基性媒体を追加し、70μmのナイロンメッシュを通して細胞を渡します。 </li><li> 1500×gで、4℃で遠心細胞懸濁​​液を5分間。 </li><li>間質細胞は現在、さらなる分析のために使用することができる。 </li></ol>リンパ節間質細胞の染色2。 <ol><li>成功したTRC、LEC、BECおよびDN細胞を認識するために抗CD45、抗ポドプラニン（GP38）、抗CD31抗体の組み合わせを使用してください。 </li><li> 2を含む100μlのHBSS中の抗GP38-PE（1：200）、抗CD31-APC（200 1）：ライブ/デッドトラッカー、抗CD45-FITC（200 1）で消化したリンパ節組織をインキュベート暗所で少なくとも20分間％のFCS、4°C、。 </li><li> 2％FCSを含むHBSSの500リットルを追加し、細胞を洗浄</li><li> 1500×gで、4℃で遠心細胞懸濁​​液を3分。 </li><li> 2％FCSを含むHBSS100μl中に再懸濁する。 </li><li> equippフローサイトメーターで染色された細胞を実行します以下の光学系とED：励起最大3つのレーザーを持つソース：青（488nmで、空冷式、20 mWのソリッドステート）、赤（633nmで、17 mWのヘリウムネオン）、バイオレット（405nmで、30 mWのソリッドステート） 。 </li><li>門CD45 -細胞は造血細胞を排除する。 </li><li>ダブレットと死細胞を除外するためのゲート·シングレット（FSC-W）と生細胞（LIVE / DEADトラッカー）。 </li><li> 、LEC（GP38 + CD31 +）、BEC（GP38 - CD31 +）およびDN（GP38 - CD31 - ）細胞（ 図1参照） - TRC（GP38 + CD31）を可視化する、CD31 対プロットGP38。 </li></ol>

<ol>
	<li>Mueller, S. N., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoid+stroma+in+the+initiation+and+control+of+immune+responses.">Lymphoid stroma in the initiation and control of immune responses.</a> Immunological reviews. 224, 284-294 (2008).</li><li>Roozendaal, R., Mebius, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+Cell+-+Immune+Cell+Interactions.">Stromal Cell - Immune Cell Interactions.</a> Annual Review of Immunology. 29 (1), 23-43 (2011).</li><li>Turley, S. J., Fletcher, A. L., Elpek, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stromal+and+haematopoietic+antigen-presenting+cells+that+reside+in+secondary+lymphoid+organs.">The stromal and haematopoietic antigen-presenting cells that reside in secondary lymphoid organs.</a> Nature Reviews Immunology. 10 (12), (2010).</li><li>Baj&#233;noff, M., 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=Stromal+cell+networks+regulate+lymphocyte+entry,+migration,+and+territoriality+in+lymph+nodes.">Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes.</a> Immunity. 25 (6), 989-1001 (2006).</li><li>Katakai, T., Hara, T., Sugai, M., Gonda, H., Shimizu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+node+fibroblastic+reticular+cells+construct+the+stromal+reticulum+via+contact+with+lymphocytes.">Lymph node fibroblastic reticular cells construct the stromal reticulum via contact with lymphocytes.</a> The Journal of experimental medicine. 200 (6), 783-795 (2004).</li><li>Link, 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=Fibroblastic+reticular+cells+in+lymph+nodes+regulate+the+homeostasis+of+naive+T+cells.">Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells.</a> Nature Immunology. 8 (11), 1255-1265 (2007).</li><li>Malhotra, D., 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=Transcriptional+profiling+of+stroma+from+inflamed+and+resting+lymph+nodes+defines+immunological+hallmarks.">Transcriptional profiling of stroma from inflamed and resting lymph nodes defines immunological hallmarks.</a> Nature Immunology. 13 (5), 499-510 (2012).</li><li>Gretz, J. E., Norbury, C. C., Anderson, A. O., Proudfoot, A. E., Shaw, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph-borne+chemokines+and+other+low+molecular+weight+molecules+reach+high+endothelial+venules+via+specialized+conduits+while+a+functional+barrier+limits+access+to+the+lymphocyte+microenvironments+in+lymph+node+cortex.">Lymph-borne chemokines and other low molecular weight molecules reach high endothelial venules via specialized conduits while a functional barrier limits access to the lymphocyte microenvironments in lymph node cortex.</a> The Journal of experimental medicine. 192 (10), 1425-1440 (2000).</li><li>Sixt, M., 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=The+conduit+system+transports+soluble+antigens+from+the+afferent+lymph+to+resident+dendritic+cells+in+the+T+cell+area+of+the+lymph+node.">The conduit system transports soluble antigens from the afferent lymph to resident dendritic cells in the T cell area of the lymph node.</a> Immunity. 22 (1), 19-29 (2005).</li><li>MartIn-Fontecha, 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=Regulation+of+dendritic+cell+migration+to+the+draining+lymph+node:+impact+on+T+lymphocyte+traffic+and+priming.">Regulation of dendritic cell migration to the draining lymph node: impact on T lymphocyte traffic and priming.</a> The Journal of experimental medicine. 198 (4), 615-621 (2003).</li><li>Luther, S. A., Tang, H. L., Hyman, P. L., Farr, A. G., Cyster, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexpression+of+the+chemokines+ELC+and+SLC+by+T+zone+stromal+cells+and+deletion+of+the+ELC+gene+in+the+plt/plt+mouse.">Coexpression of the chemokines ELC and SLC by T zone stromal cells and deletion of the ELC gene in the plt/plt mouse.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (23), 12694-12699 (2000).</li><li>Braun, 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=Afferent+lymph-derived+T+cells+and+DCs+use+different+chemokine+receptor+CCR7-dependent+routes+for+entry+into+the+lymph+node+and+intranodal+migration.">Afferent lymph-derived T cells and DCs use different chemokine receptor CCR7-dependent routes for entry into the lymph node and intranodal migration.</a> Nature Immunology. 12 (9), 879-887 (2011).</li><li>Fletcher, A. 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=Reproducible+isolation+of+lymph+node+stromal+cells+reveals+site-dependent+differences+in+fibroblastic+reticular+cells.">Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells.</a> Frontiers in immunology. 2, 35 (2011).</li><li>Lukacs-Kornek, 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=Regulated+release+of+nitric+oxide+by+nonhematopoietic+stroma+controls+expansion+of+the+activated+T+cell+pool+in+lymph+nodes.">Regulated release of nitric oxide by nonhematopoietic stroma controls expansion of the activated T cell pool in lymph nodes.</a> Nature Immunology. 12 (11), 1096-1104 (2011).</li><li>Siegert, S., 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=Fibroblastic+reticular+cells+from+lymph+nodes+attenuate+T+cell+expansion+by+producing+nitric+oxide.">Fibroblastic reticular cells from lymph nodes attenuate T cell expansion by producing nitric oxide.</a> PLoS ONE. 6 (11), e27618 (2011).</li><li>Khan, O., Headley, M., Gerard, A., Wei, W., Liu, L., Krummel, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+T+cell+priming+by+lymphoid+stroma.">Regulation of T cell priming by lymphoid stroma.</a> PLoS ONE. 6 (11), e26138 (2011).</li><li>Svensson, M., Maroof, A., Ato, M., Kaye, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cells+direct+local+differentiation+of+regulatory+dendritic+cells.">Stromal cells direct local differentiation of regulatory dendritic cells.</a> Immunity. 21 (6), 805-816 (2004).</li><li>Onder, 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=Endothelial+cell-specific+lymphotoxin-&#946;+receptor+signaling+is+critical+for+lymph+node+and+high+endothelial+venule+formation.">Endothelial cell-specific lymphotoxin-&#946; receptor signaling is critical for lymph node and high endothelial venule formation.</a> The Journal of experimental medicine. 210 (3), 465-473 (2013).</li><li>Fletcher, A. 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=Lymph+node+fibroblastic+reticular+cells+directly+present+peripheral+tissue+antigen+under+steady-state+and+inflammatory+conditions.">Lymph node fibroblastic reticular cells directly present peripheral tissue antigen under steady-state and inflammatory conditions.</a> The Journal of experimental medicine. 207 (4), 689-697 (2010).</li><li>Fletcher, A. L., Malhotra, D., Turley, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+node+stroma+broaden+the+peripheral+tolerance+paradigm.">Lymph node stroma broaden the peripheral tolerance paradigm.</a> Trends in Immunology. 32 (1), 12-18 (2011).</li><li>Cohen, J. N., 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=Lymph+node-resident+lymphatic+endothelial+cells+mediate+peripheral+tolerance+via+Aire-independent+direct+antigen+presentation.">Lymph node-resident lymphatic endothelial cells mediate peripheral tolerance via Aire-independent direct antigen presentation.</a> Journal of Experimental Medicine. 207 (4), 681-688 (2010).</li><li>Lee, J. -. W., 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=Peripheral+antigen+display+by+lymph+node+stroma+promotes+T+cell+tolerance+to+intestinal+self.">Peripheral antigen display by lymph node stroma promotes T cell tolerance to intestinal self.</a> Nat Immunol. 8, 181-190 (2006).</li><li>Nichols, L. 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=Deletional+self-tolerance+to+a+melanocyte/melanoma+antigen+derived+from+tyrosinase+is+mediated+by+a+radio-resistant+cell+in+peripheral+and+mesenteric+lymph+nodes.">Deletional self-tolerance to a melanocyte/melanoma antigen derived from tyrosinase is mediated by a radio-resistant cell in peripheral and mesenteric lymph nodes.</a> J Immunol. 179, 993-1003 (2007).</li><li>Gardner, J. M., 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=Deletional+tolerance+mediated+by+extrathymic+Aire-expressing+cells.">Deletional tolerance mediated by extrathymic Aire-expressing cells.</a> Science. 321, 843-847 (2008).</li><li>Gardner, J. M., 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=Extrathymic+Aire-expressing+cells+are+a+distinct+bone+marrow-derived+population+that+induce+functional+inactivation+of+CD4++T+cells.">Extrathymic Aire-expressing cells are a distinct bone marrow-derived population that induce functional inactivation of CD4+ T cells.</a> Immunity. 39, 560-572 (2013).</li><li>Magnusson, F. C., 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=Direct+presentation+of+antigen+by+lymph+node+stromal+cells+protects+against+CD8+T-cell-mediated+intestinal+autoimmunity.">Direct presentation of antigen by lymph node stromal cells protects against CD8 T-cell-mediated intestinal autoimmunity.</a> Gastroenterology. 134, 1028-1037 (2008).</li><li>Tewalt, E. 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=Lymphatic+endothelial+cells+induce+tolerance+via+PD-L1+and+lack+of+costimulation+leading+to+high-level+PD-1+expression+on+CD8+T+cells.">Lymphatic endothelial cells induce tolerance via PD-L1 and lack of costimulation leading to high-level PD-1 expression on CD8 T cells.</a> Blood. 120 (24), 4772-4782 (2012).</li></ol>

The authors declare they have no competing financial interests.

リンパ節間質細胞の研究は、最近公開された消化による2プロトコル6,13の開発に研究の焦点となった。両方のプロトコルは、単一のリンパ節間質細胞を獲得するのに十分であるが、消化酵素と消化の時間の使い方が異なります。間質細胞およびそれらの表面マーカーは、酵素消化および機械的応力に敏感であるため、最適化されたプロトコルが必要とされる。 新鮮に解剖し、リンパ節から実行可能なリンパ節間質細胞の単離は、表現型および機能解析を実行するための最初のステップです。したがって、慎重に安定した温度及び示された時間のために、酵素の規定された濃度で消化することが重要である。二つ消化プロトコルがしかしかなり長い消化ステップで、6,13に記載されている。消化の時間を短縮するために、機械的な分解が含まれ、マルチチャネルピペットを用いて標準化した。上の<html>現在のプロトコルの利点の電子はしたがって、消化酵素とのインキュベーション時間を最小限に、わずか45分で間質細胞の単離を可能にすることである。また、消化中の一定の撹拌は、リンパ節構造への消化酵素の最適なアクセスを可能にする。また、消化容積は、使用する酵素の量を最小μlの750に減少した。 過度の機械的な力は、タイトジャンクショ​​ンを破壊が、同時に細胞死を引き起こす可能性があります。このような理由から、私たちは再懸濁サイクルと異なる解離デバイスのいくつかの組み合わせをテストした。私たちは、自動化されたマルチチャンネルピペットの使用は、99サイクルの2アプリケーション内で解離を可能にすることを見出した。細胞に一定の圧力を適用し、これが最適な生存能力を有する単一の細胞懸濁液をもたらす。 4〜6個の試料を混合し、リンパ節間質細胞単離の時間を低減すると同時にピペッティングすることができる。 ontent &quot;&gt;消化プロトコルは、ほとんどすべての分子の表面発現を維持しなければならない;集団は、均一または不均一である場合にのみ、このようにいくつかのマーカーとの併用染色を明らかにすることができ、例えば、中BP-3およびCD35の使用。 TRCのGP38 + CD31 - 。髄TRC（Sanjivルター、私信）の可視化および単離が可能になりさらに、特徴付けが乏しいDN画分を、さらに7で検討インテグリンα7（、陽性の細胞のようなアイレ+細胞および周皮細胞を含むものとして特徴付けられた13）。従って、本プロトコルは、公開されたものと比較した。公開のものと比較して、現在のプロトコルを用いて得られた結果は、同様の周波数およびリンパ節間質細胞の数に示すように、単離されたTRCの興味深いことに、頻度および数最高現在のプロトコルに比べて、TRC構造のより良い解離を示唆しているフレッチャープロトコルを使用していたし、リンクプロトコル。さらに、さまざまな表面分子の発現は、フレッチャープロトコルに比べて現在のプロトコルで増強された。消化法におけるこれらの差異は、リンパ節間質細胞研究6,19,21においてわずかに異なる発現パターンの理由かもしれない。それが原因各プロトコルの利点の異なる消化プロトコルをテストするために役に立つかもしれません。フレッチャープロトコルは転写解析のための実行可能な細胞の高い数値を単離するために有用であるかもしれないが（マルホトラら 7に掲載されるように）現在のプロトコルは、表面発現解析のためのより良いかもしれません。したがって、すべての3つの消化プロトコルを比較するリンパ節間質細胞の単離後に最適な結果を得るために重要であり得ると相補的であると見なされるべきである。

リンパ節は、外国と自己抗原に対する適応免疫応答が開始され、調整されている特殊なコンパートメントである。ここに提示手順は、リンパ節単一細胞懸濁液を得て、いくつかの分子の表面発現を維持する実行可能な節間質細胞へのアクセスを得るために自動化された機械的ピペッティングと組み合わせる短い酵素消化を記載している。 リンパ節間質細胞、リンパ節の足場を形成し、三つの主要な機能を果たす：最初それらは存在する抗原、病原体およびそれらの病原体関連分子パターン（PAMPに）、ならびにサイトカインおよび危険性関連分子パターン（DAMPS）をサンプリングするために体液をフィルタリング体内で。第二に、それらが相互作用し、適応免疫応答を開始するための抗原提示細胞（APC）およびリンパ球を誘引し、指示する。第三に、それらは、リンパ球1の恒常性および分化のための構造的な環境を提供する-3。炎症リンパ節間質細胞は、成長因子、サイトカイン及びケモカインを産生中に、樹状細胞（DC）との相互作用を整理することにより、T-およびB細胞膨潤に適応する。免疫応答のオーケストレーションは、異なる間質細胞集団によって形成される複合体の構造的アーキテクチャにのみ可能である。 リンパ節間質細胞は、CD45陰性細胞で、線維芽細胞および内皮細胞においてCD31の発現またはGP38によって1-6を区別することができる。 GP38 + CD31が- （もFRCとしても知られ、TRC、：線維芽細胞細網細胞）、Tゾーンの網状細胞を定義して、GP38 + CD31 +がリンパ管内皮細胞（LEC）を定義して、GP38 - CD31 +は、血液内皮細胞（BEC）を定義します。さらに、集団の特徴付けは、他のリンパ節間質細胞の存在が明らかになった。実際、小周皮細胞様細胞集団内で特徴づけられたGP38 - CD31 -人口7。したがって、単離手順の適応は、異なるリンパ節間質細胞の機能的特性の同定および特徴付けのために有利である。 リンパ節間質細胞消化の開発の前にリンパ節間質細胞の研究は、組織切片や顕微鏡を用いてその場観察にに限られていたプロトコル。それにもかかわらず、構造的および機能的研究は、リンパ節間質細胞の重要な特徴を示した。リンパ節間質細胞は、高内皮へのリンパ節の被膜下洞からリンパおよび関連する低分子量タンパク質を輸送する導管システムと呼ばれる複雑な3次元構造を形成するポドプラニン、コラーゲンおよび細胞外マトリックス（ECM）タンパク質と関連しているT細胞ゾーン8に静脈。 DCは、間質細胞と密接に接触している管状詐欺に突出観察することができるduit構造は、流体サンプルと抗原8を検出する。 DCとリンパ節間質細胞（TRCはとのLEC）の相互作用はケモカインCCL21とCCL19 9,10の放出および提示によって媒介される。 CCL19およびCCL21は、リンパ節のT細胞領域4,11に移行するDCおよびT細胞を促進するCCR7受容体によって認識される。類似したケモカインを使用しているにもかかわらず、DCとT細胞は、リンパ節12内に異なった移動ルートを持っている。その後、リンパ節および純粋なリンパ節間質細胞の単離の酵素消化を使用して、機能的研究は、異なるリンパ節間質細胞の役割とDCおよびT / B細胞6,13と相互作用するその能力に基づいて行った。まず、IFN-γ産生エフェクターT細胞及びリンパ節間質細胞間のクロストークは、二次リンパ器官14-16にT細胞応答および増殖を弱めることが示さ代謝産物一酸化窒素の産生を誘導する。第二に、リンパnはオード間質細胞は、IL-10の産17を介して規制DCサブセットの分化を支持すること、およびIL-7 6,18の産生を介してナイーブT細胞の恒常性を調節することが報告されている。第三に、リンパ節間質細胞におけるTLRの発現は、間質細胞は、組織傷害において放出、感染または自己分子に由来する信号の影響を受けやすいことを示唆している。実際に、TLR3ポリリガンドとリンパ節間質細胞の処理は、（I：C）は、結果として共阻害分子PD-L1のではなく、共刺激分子の主要組織適合性複合体クラスIの発現及び上方制御の控えめな上方制御を誘導する末梢組織抗原発現の19の劇的な変化。いくつかのグループが、リンパ節間質細胞は、末梢組織抗原を発現し、自己反応性T細胞19,21-27の寛容を誘導することが示されている。したがって、リンパ節間質細胞および他の遊走および再間の相互作用を理解することsidentリンパ節細胞は、炎症の間に活性化または免疫応答の抑制を可能にする新たな標的分子を見つけるために役立つ。したがって、リンパ節の公開酵素分離の実施が必要である。 以前公開されたプロトコルは、低機械的ストレス6,19,20とコラゲナーゼベースの酵素消化の異なる組み合わせを使用しています。しかし、消化酵素または消化酵素の異なる組み合わせの長いインキュベーションは、活性化状態を分析するために、新たなリンパ節間質細胞を同定するために必要な各種表面分子を劣化させるかもしれない。間質細胞分析の種類に応じて、リンクプロトコルまたはフレッチャー·プロトコルは、より適しているかもしれません。記載された手順では、わずかに短い酵素消化は、実行可能なリンパ節間質細胞の表面マーカーの劣化を最小限にするために自動化された機械的解離と組み合わされる。この手順では、再現性の高い分離を可能にし、低い変動性と95％以上の生存率を有するリンパ節間質細胞集団の区別。新たに単離したリンパ節間質細胞は、 インビトロで直接機能アッセイを実行するために、表面マーカー発現、タンパク質分析、および転写の研究、ならびに間質細胞株の樹立のために使用することができる。

<table border="0" cellpadding="0" cellspacing="0" width="959">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:231px;">LifeTechnologies-Gibco</td>
			<td style="width:119px;">41965-039</td>
			<td style="width:395px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FCS</td>
			<td style="width:231px;"></td>
			<td style="width:119px;"></td>
			<td>Final concentration 2%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:119px;">499609</td>
			<td>Final concentration 1.2 mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Collagenase IV</td>
			<td style="width:231px;">Worthington Biochemical Corporation</td>
			<td style="width:119px;"></td>
			<td>&#62;160 units per mg dry weight, use at final concentration of 1 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase D</td>
			<td style="width:231px;">Roche</td>
			<td style="width:119px;">11088882001</td>
			<td>use at 3.5 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNAse I</td>
			<td style="width:231px;">Roche&#160;</td>
			<td style="width:119px;">11284932001</td>
			<td>use at 40 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">stiring magnets</td>
			<td style="width:231px;">FAUST</td>
			<td style="width:119px;"></td>
			<td>5 mm long-2 mm &#248;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polystyren Round-Bottom Tubes 5ml</td>
			<td style="width:231px;">Falcon-BD Bioscience</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Magnetic stirrer with heating funktion</td>
			<td style="width:231px;">IKA-RCT-standard</td>
			<td style="width:119px;">9720250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petridishes 100 mm, sterile</td>
			<td style="width:231px;">TPP</td>
			<td style="width:119px;">6223201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25G needles</td>
			<td style="width:231px;">Terumo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD45 Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 30-F11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD11c Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone N418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse Podoplanin Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 8.1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD31 Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone MEC13.3</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;"> 
			Eppendorf Xplorer plus, Multichannel</td>
			<td style="width:231px;">Eppendorf</td>
			<td style="width:119px;">4861 000.821/830</td>
			<td>1.250 &#181;l max. volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD140a</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone APA5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD80</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 16-10A1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD40</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 1C10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse I-Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone AF6-120.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD274 (PD-L1)</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 10F.9G2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LIVE/DEAD Fixable Near-IR Dead Cell stain kit</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:119px;">L10119</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

現在のプロトコルが原因自動化されたマルチチャンネルピ ​​ペットで機械的に解離に短い消化時間（45分以内）にリンクら 、2007 6によって公開修正された消化プロトコルです。また、手順はより標準化され、異なるリンパ節間質細胞上の表面マーカーの劣化を最小限に抑え、同時に複数のサンプルの処理を可能にする。 コラゲナーゼIVおよびコラゲナーゼDリンクプロトコル6と現在のプロトコルまたはコラゲナーゼP内およびディスパーゼFletchersプロトコルでの13リンパ節間質細胞の生存率を維持し、CD45、GP38およびCD31を含むいくつかの表面マーカーの消化を惜しま。テストするために現在のプロトコル、腋窩、上腕および鼠径リンパ節を取り出し、それらの間質細胞を単離するために消化した。 CD45、GP38およびCD31発現の分析を、C57BL / 6メートルから分離されたTRC、LEC、BECおよびDN細胞の存在を実証した氷（ 図1、ゲート戦略）。 機械的ストレスが消化中リンパ節間質細胞の生存率を減少させるかもしれない。三つの異なるプロトコルを使用して、間質細胞の単離集団の生存率を比較すると、それは現在のプロトコルはより良い表示が、生存率は、現在のプロトコルとリンクプロトコル（ 図2A）の両方にフレッチャープロトコルにおける95％よりも高いが、より低いことがわかったBEC亜集団での生存率は、プロトコルをリンクします。全細胞数は、リンパ節間質細胞サブセットのポスト消化を回収するリンクプロトコルおよびフレッチャープロトコル（ 図2A）と比較して現在のプロトコルでわずかに高かった。これらの結果は、現在のプロトコルは、公開されたプロトコルに類似するリンパ節間質細胞の生存能力を維持することを実証する。 3つのプロトコルの主な違いを表1に記載されています。 表面マーカーaを<html>フローサイトメトリーによるリンパ節間質細胞を特徴付けるために、セルソーティングによってそれらを分離するために必要な再。 TRCはとLECが、リンクおよびフレッチャープロトコルはIA b を 、CD140a、CD80、PD-L1およびCD40発現のために現在を比較した孤立ビア。すべての3つのプロトコルにより間質細胞を単離した後、私たちは、IA b を 、CD80、CD140a、PD-L1の発現を発見し、CD40はTRCをとのLEC（ 図2B）の両方でのCPおよびLPでの消化の際に高かった。これらの結果は、コラゲナーゼIVおよびDは、いくつかの表面分子の劣化がコラゲナーゼPおよびディスパーゼの場合ほど強​​いことを示唆している。 まとめると、現在のプロトコルは、生存リンパ節間質細胞の表面マーカーの劣化を最小限にするために自動化された機械的脱凝集と組み合わせた短い消化を含む。 &quot;/&gt; C57BL / 6マウスから、図1のTRC、LEC、BEC、およびDN細胞の染色、ゲーティング、および定量。リンパ節は、現在のプロトコルに従って消化し ​​、TRC、LECを定義するために、CD45、GP38およびCD31、/ライブデッドで染色したフローサイトメトリーによるBEC、およびDN細胞。データは、染色の代表的な結果を示しています。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51803/51803fig1highres.jpg" target="_blank">大きな画像を見るにはここをクリックしてください。</a> <img alt="図2" fo:content-width="5in" src="/files/ftp_upload/51803/51803fig2highres.jpg" width="500" /> リンパ節間質細胞の図2。生存率および表面マーカー発現。C57BL / 6マウスからのリンパ節は、Linkの（LP）とフレッチャーのプロトコル（FP）とCD45、GP38およびCD31で染色し、定義する電流（CP）は、次の消化し ​​たフローサイトメトリーによるTRC、LEC、BECおよびDN細胞。A）Viabil性と生/死染色後、すべてのリンパ節間質細胞集団の総細胞数。B）IA b の表面発現の分析（APC-Cy7で）、CD140a（nは≥5、2つの独立した実験のプールされたデータが、バーはSEMを表す） （PerCPを-Cy5.5の）、CD80（PerCPを-Cy5.5の）、PD-L1（PE-Cy7で）とTRCを上のCD40（PE-Cy7は）とのLECた（n = 6、LP及びCPのためのFPであり、n = 11 、2つの独立した実験、バーはSEMを表す）のデータをプールした。 LEC-APC-Cy7での幾何MFIの自家蛍光値：324±97、LEC-PECy7：163±82.5、LEC-PerCpCy5.5：156±36、TRC-APC-Cy7で：349±152、TRC-PECy7：117 121±45：TRC-PerCPを-Cy5.5のは、54±。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51803/51803fig2highres.jpg" target="_blank">大きな画像を見るにはここをクリックしてください。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>現在のプロトコル、酵素</td><td>現在のプロトコル</td><td>リンクプロトコル酵素</td><td>リンク·プロトコル</td><td> Fレッチャープロトコル酵素</td><td>フレッチャー·プロトコル</td></tr><tr><td>コラゲナーゼIV、DNアーゼI </td><td> 30分、37℃、攪拌</td><td>コラゲナーゼIV、DNアーゼI </td><td> 30分間、37°C </td><td>コラゲナーゼP、ディスパーゼ、DNアーゼI </td><td> 20分、37°C​​で、5分毎に反転</td></tr><tr><td>コラゲナーゼD、DNアーゼI </td><td> 5分、37℃で、撹拌、再懸濁</td><td>コラゲナーゼD、DNアーゼI </td><td> 20分、37°C </td><td>コラゲナーゼP、ディスパーゼ、DNアーゼI </td><td> 10分間、37°C​​で、30秒間再懸濁</td></tr><tr><td></td><td> 10分、37℃、攪拌</td><td>コラゲナーゼD、DNアーゼI </td><td> 37℃、完全消化するまで10分ごとに再懸濁</td><td>コラゲナーゼP、ディスパーゼ、DNアーゼI </td><td> 37℃、完全消化するまで5分毎に再懸濁</td></tr><tr><td></td><td>自動化された機械的な再懸濁</td><td></td><td></td><td></td><td></td></tr></tbody></table> 表のCP、LPの1短い要約、FPの使用される酵素とプロトコル。

Watch this Scientific Journal Video about Isolation of Murine Lymph Node Stromal Cells at JoVE.com

Isolation of Murine Lymph Node Stromal Cells

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 ...

isolation-of-murine-lymph-node-stromal-cells

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

31.0K ビュー.  University of Basel and University Hospital Basel. 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. 

ビデオ: Isolation of Murine Lymph Node Stromal Cells

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 ...

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 ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

マウス歯肉細胞の単離、処理と解析

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

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, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

単離、同定、およびマウス胸腺上皮細胞の精製

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

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 ...

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, ...