この作品は、（KAH）は健康グラントR01 AI088209の国立研究所によってサポートされていました。また、ミネソタフローサイトメトリーのリソースの大学に感謝します。

本研究では、成人（6 - 8週）の雌のC57Bl / 6マウスを用いた。マウスは、国立癌研究所から購入し、特定の病原体フリーの条件下で維持した。ミネソタ大学の施設内動物管理使用委員会（IACUC）は、すべての動物実験を承認した。 楽器とバッファの調製<ol><li>酵素液の準備、アルブミンが豊富なバッファー（1×PBSの[Ca 2 + / Mgの2+を含まない]と0.5％ウシ血清（0.05％RPMI1640培地をリベラーゼTH及びDNアーゼIの100単位/ mlの[w / vの]）アルブミン及び2mMエチレンジアミン四酢酸[EDTA]）、FACS緩衝液（1×PBSの[Ca 2 + / Mgの2 +を含まない]を含む1％ウシ胎児血清[FBS]、0.02％のNaN 3）、FACSバッファソーティング（1X PBS 10％FBSが供給された[Ca 2 + / Mgが2 +を含まない]、2％FBS）を含有する、およびRP10培地（RPMI-1640培地）。 </li><li> 10パニングプレートを準備します。 （0.01 mg / mlのGを40ml塗布液を調製しオート麦は、0.1MのNaHCO 3緩衝液中の抗ラットIgG）を、それぞれ100×15ミリメートルプレート、スワールに塗布液の4ミリリットルを追加し、4℃、CO / Nインキュベートする。抗体溶液を捨て2回1×5mlのPBSを用いてプレートをすすぎ、リンスの間で活発に渦巻く。 4℃で準備されたプレートを保管してください。 </li></ol>胸腺組織の2収穫<ol><li> CO 2チャンバー内でマウスを安楽死させる。 、解剖マットの上に仰向けに動物を入れてマットの上に足をピンと70％エタノールを噴霧して毛皮を濡らす。 </li><li>まっすぐにハサミで下顎への尿道口の上から出発して、皮膚を​​介して正中切開を行い、両側の膝に下向きに切開を延長。切開は「Y」逆さまのように見えます。両側のたるんだ皮膚を後ろに引き、そして解剖マットにそれをピン。 </li><li>次いで、肋骨を持ち上げ、鎖骨付近までそれぞれの側にリブをカット、剣状突起から横隔膜を穿刺鉗子は解剖マットにそれをピンと。胸腺はちょうど肋骨の下にあり、心の上にある二つの薄い白のローブ（ 図1）のように見えます。胸腺の周囲の結合組織を外し、緩やかに湾曲鋸歯状の鉗子で胸腺ローブのペアを引っ張って外します。 5ミリリットルのRPMI-1640を含む6ウェルプレートに胸腺葉を置きます。 </li></ol>胸腺間質細胞の調製<ol><li>周囲の脂肪と結合組織をトリミングし、新たに酵素液を調製5ミリリットルを含む6ウェルプレートの新しいウェルに胸腺葉を移す。細かいハサミで胸腺葉に小さな切開を行い、20分間、37℃でプレートをインキュベートする。 </li><li>組織解離のための5ミリリットルピペットを用いて3回 - 静かにピペットを上下に2を胸腺組織を消化。 10ミリリットルの酵素を中和するコールドアルブミンが豊富な緩衝液を含む50mlチューブに上清画分を収集する。氷上でコレクションチューブを保管してください。 2.5を追加します。残りの組織への溶解酵素溶液と15分間37℃でプレートをインキュベートする。 </li><li>凝集体を破壊するために3ミリリットルの注射器と、18G針で穏やかな機械的撹拌を実行します。コレクションチューブにプール上清。残りの組織に2.5ミリリットルの酵素溶液を加え、15分間、37℃でプレートをインキュベートする。 </li><li> 3ミリリットルの注射器と25 Gの針で穏やかな機械的撹拌を繰り返し、プレートをさらに5インキュベート - 10分。 </li><li>完全消化した後、コレクションチューブに上清をプールする。遠心分離した細胞を収集するために8分間400×gで、4℃でコレクションチューブ。液体を吸引し、10mlのアルブミンが豊富なバッファ内のペレット化した細胞を中断し、100mmのメッシュを通してサンプルをフィルタリングする。血球計数器を用いて細胞を数える。その後、フローサイトメトリー分析（第4節）、またはTECの濃縮（第5節）を行う。 </li></ol>フローサイトメトリーによるのTECの4の同定</p&gt; <ol><li> 96ウェル丸底試験板に染色FACS緩衝液中に100μlあたり4×10 6細胞の最終濃度に細胞を調製し、氷上にプレートを保つ。細胞に（FACS緩衝液中10μg/ mlの抗CD16 / CD32抗体）を20μlのFcブロック溶液を加え、氷上で10分間細胞をインキュベートします。 400×gで、3分間、4℃でプレートをスピン。流しにダウン板面をフリックでウェルから液体を捨ててください。 </li><li> 100μlの抗体カクテル（抗CD45、-EpCAM、-MHCクラスII、および-Ly51抗体におけるペレット化した細胞を一時停止し、メーカーで異なる蛍光色素で標識したUEA-1レクチンは、FACSバッファ内の各抗体の最適な濃度をお勧めしますまたはテスト）し、暗所で20分間氷上で細胞をインキュベートする。補償のために、各個別の蛍光色素で1×10 6個の細胞を染色する。 </li><li>各ウェルにFACS緩衝液100μlを加え、400×gで、3分間、4℃でプレートをスピン。洗浄ステップ1を繰り返します時間、その後100μlのFACS緩衝液中で細胞を再懸濁。 300μlのFACS緩衝液で満たされた12×15mmの丸底FACSチューブに細胞を移す。フローサイトメーターでサンプルを取得し、FACS分析ソフトウェアを用いてデータを分析する。 TECゲーティング戦略は、 図2に示されている。 </li></ol>パンニングによるTECの5。エンリッチメント<ol><li> 5分間400×gで、4℃で（セクション3で調製した）胸腺細胞をスピンダウン。吸引した液体と15ミリリットルまたは50 mlコニカルチューブ中の1ml当たり2×10 8細胞の濃度でFACSソーティングバッファ内のペレット化した細胞を一時停止する。細胞懸濁液を2.5μg/ mlの最終濃度で抗CD90.2抗体（クローン30-H12）（または抗CD45）を追加し、30分間4℃で回転ミキサー上で穏やかに細胞をインキュベートする。インキュベーションに続いて、5で洗浄 - RP10培地の10倍の体積と400×gで、5分間、4℃でチューブを遠心する。 </li><li>吸引した液体とでペレット化した細胞を再懸濁1mlあたり1×10 7の濃度でRP10培地。各コーティングされたパン（ステップ1.3で調製）プレート、スワール5ミリリットルの細胞懸濁液を加え、室温で30分間インキュベートする。精力的に渦巻く;コニカルチューブに上清を移す。プレートをRP10培地を用いて2回すすぎ、収集チューブに上清をプールする。 </li><li> 400×gで、5分間、4℃でコレクションチューブを遠心。吸引した液体と5ミリリットルのRP10培地中でペレット化した細胞を懸濁します。 、新しいパンプレートに細胞懸濁液を追加旋回し、室温で30分間インキュベートする。細胞がプレートをすすぎ、コニカルチューブにそれらをプール収集します。 TECのが濃縮されると、5分間、4℃、400×gで細胞をスピンダウンし、バッファをソートする5ミリリットルのFACSで細胞を一時停止。 </li></ol>細胞選別によるのTECの6。精製<ol><li>血球計数器を用いて細胞を数え、FACSソーティングバッファ内の4×10 7細胞の濃度で細胞懸濁液を準備します。抗体デスを追加細胞溶液に4.4節でのcribed、30分間、4℃で回転ミキサー上で穏やかに細胞を培養する。染色の後、洗浄し、FACSソーティングバッファの1mlあたり10×10 6細胞の濃度に細胞を懸濁する。 </li><li>蛍光活性化セルソーティングを使用して、100μMのノズルを通してソートテックサブセット。ソートされた細胞は、RPMI-1640中30％（v / v）のFBS）に集められる。細胞選別は、5分間、400×gで、4℃で遠心分離を行って細胞および下流の分析のために準備される一度。 </li></ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractionation+of+T+cells+and+B+cells+using+panning+techniques.">Fractionation of T cells and B cells using panning techniques.</a> Curr. Protoc. Immunol. 3 (Unit 3.5), (1991).</li><li>Wekerle, H., Ketelsen, U. P., Ernst, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymic+nurse+cells.+Lymphoepithelial+cell+complexes+in+murine+thymuses:+morphological+and+serological+characterization.">Thymic nurse cells. Lymphoepithelial cell complexes in murine thymuses: morphological and serological characterization.</a> The Journal of experimental medicin. 151, 925-944 (1980).</li><li>Kyewski, B. A., Kaplan, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoepithelial+interactions+in+the+mouse+thymus:+phenotypic+and+kinetic+studies+on+thymic+nurse+cells.">Lymphoepithelial interactions in the mouse thymus: phenotypic and kinetic studies on thymic nurse cells.</a> J Immuno. 128, 2287-2294 (1982).</li><li>Nakagawa, Y., 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=Thymic+nurse+cells+provide+microenvironment+for+secondary+T+cell+receptor+alpha+rearrangement+in+cortical+thymocytes.">Thymic nurse cells provide microenvironment for secondary T cell receptor alpha rearrangement in cortical thymocytes.</a> Proc. Natl. Acad. Sci. U. S. A. 109, 20572-20577 (2012).</li><li>McLelland, B. T., Gravano, D., Castillo, J., Montoy, S., Manilay, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+isolation+of+adult+thymic+epithelial+cell+subsets+for+multiparameter+flow+cytometry+and+gene+expression+analysis.">Enhanced isolation of adult thymic epithelial cell subsets for multiparameter flow cytometry and gene expression analysis.</a> J. Immunol. Method. 367, 85-94 (2011).</li><li>Seach, N., Wong, K., Hammett, M., Boyd, R. L., Chidgey, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purified+enzymes+improve+isolation+and+characterization+of+the+adult+thymic+epithelium.">Purified enzymes improve isolation and characterization of the adult thymic epithelium.</a> Journal of immunological method. 385, 23-34 (2012).</li></ol>

著者は、彼らが競合する金融利害がないことを宣言します。

プロトコルでは、重要なステップは、胸腺間質細胞（第3節）とのTECの濃縮（第4節）の製造である。ことを強く新鮮な酵素溶液を毎回用意し、組織をできるだけ早く扱われることをお勧めします。プールされた胸腺、酵素溶液の容量を最適化する胸腺の数に応じて必要とされる。任意の組織残留物は、処理後に残っている場合、より多くの酵素溶液及びインキュベーション時間の延長を添加?...

初期のT細胞の開発で、骨髄の造血幹細胞由来の多能性前駆細胞が胸腺の皮質に補充される、T細胞系列へのコミットメントを受け、T細胞前駆体1になる。非常に変化したT細胞受容体1と前駆細胞の大きなプールを形成し、皮質では、T細胞前駆体のCD4とCD8ダブルネガティブ（DN）胸腺細胞が膨張し、未成熟CD4およびCD8ダブルポジティブ（DP）胸腺細胞に分化する。 DP細胞の選択MHC拘束性サブセットは、CD4またはCD8シングルポジティブ（SP）胸腺細胞になる胸腺の髄質に移行し、機能的に有能な成熟T細胞へのような正の選択2-と呼ばれるイベントを差別化するだけ6。これとは対照的に、自己反応性胸腺細胞のクローンは、負の選択を受け、自己寛容のための制御性T細胞に変換するか、まだ明らかでない目的のために上皮内リンパ球に転用、アポトーシスによって取り除かれる3,7-10。 胸腺は、胸腺間質細胞は、これらの多様なT細胞の発生運命5,11,12のための信号を提供するユニークな微小環境を形成している。皮質のTEC（cTECs）および髄質のTEC（mTECs）、樹状細胞、マクロファージ、線維芽細胞、内皮細胞、神経堤由来の周皮細胞および他の間葉系細胞を13〜15を含む-胸腺間質細胞は、胸腺上皮細胞（TECの）から構成されている。これらの中でも、TECは、T細胞の発達1,2,16,17のさまざまな段階で重要である。しかし、TECのを隔離するための確実な方法がないことは、それらの機能16の包括的な理解を妨げている。特に、皮質の前駆細胞を囲む三次元網目構造を形成cTECsは、まだ明らかでない理由のために正の選択13,18,19のために必須である。初期の研究は、主に形態学的および組織学的ツール13に頼ること、のTECの不均一性や役割などの手掛かりを提供</suP&gt;。最近のTECサブセットのユニークな役割は、マウスモデル12,20における遺伝的アプローチによって対処された。 TECのを単離するための堅牢で再現性のある方法がcTECsが正の選択をサポートする方法のTECサブセットの公正な特性評価、TEC機能の定量的および定性的評価、およびメカニズムの解明を達成することが基本である。 ため、彼らは無傷の器官において形成胸腺内のTECの希少性とタイトな相互作用のために、TECはの単離は困難なされています。ここで説明するプロトコルは、以前の発見、現在利用可能な試薬、技術、および胸腺構造および間質組成物の知識に基づいている。ほぼ20年前に、いくつかの手順は、トリプシン、コラゲナーゼとディスパーゼを含むさまざまな酵素が消化中に使用された胸腺組織21-27を 、脱凝集することが報告された。 Gray ら 。彼らprecedure 28のものの酵素を比較し、改善されたメタを報告した広く使用されている20,30になった酵素カクテル29の多段消化し ​​たOD。しかし、この方法は、長い準備時間と同じでもマウスのコホート29,30でのTECの可変の最終細胞数および割合で複雑 ​​な消化工程および結果を含む。数年前、リベラーゼ研究グレード酵素、高度にコラゲナーゼを精製し、中性プロテアーゼは、胸腺組織解離30で使用され始め含有する。ここでは、マウスの胸腺組織からの生のTECの高い数を出し最適化された機械的分離手順リベ消化ベースのプロトコルを記載している。 。 解離した間質細胞を濃縮するために、以前の研究では、密度勾配または磁気ビーズ分離21,29のいずれかを使用している。しかし、両方の方法は、胸腺ストローマ細胞、cTECs 29の特に人口の特定の集団から失わ厳しい引き起こす。細胞傷害性除去とパニング技術<s&gt; 31,32まで広く免疫学分野12,31に枯渇またはリンパ球を分離するために使用されている。これらの技術の比較後、私たちはTECのを濃縮するための現在のパン·プロトコルを確立した。濃縮手順の間に緩やかな条件が少ない細胞死および公正な増加TECの回復につながる。 セクション3に記載される単離され胸腺細胞懸濁液を直接TECサブセット及び樹状細胞の同定および特徴付けのためにフローサイトメトリー分析に適用することができる。第4節では、サイトメーター多パラメーターフローを使用して、TECのサブセットを識別するための簡単​​で便利な方法を説明しています。精製されたcTECsまたはmTECs、TEC濃縮と手順をセルソーティングを得ようとした実験については、セクション5と6に記載されています。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:487px;">Anti-mouse EpCAM</td>
			<td style="width:153px;">eBioscience</td>
			<td style="width:133px;">XX-5791-YY*</td>
			<td style="width:163px;">Clone G8.8</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-mouse MHC II</td>
			<td>eBioscience</td>
			<td>XX-5321-YY*</td>
			<td>Clone M5/114.15.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-mouse Ly51</td>
			<td>eBioscience</td>
			<td>XX-5891-YY*</td>
			<td>Clone 6C3</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UEA-1</td>
			<td>Vector Laboratory</td>
			<td>FL-1061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cytometer</td>
			<td>BD Biosciences&#160;</td>
			<td>LSRFortessa</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cell sorter</td>
			<td>BD Biosciences&#160;</td>
			<td>FACSAria</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACS tubes</td>
			<td>BD Biosciences</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flow cytometry analysis software</td>
			<td>TreeStar - Flowjo</td>
			<td>FlowJo v7/9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">*XX varies by fluorochrome and YY varies by vial size.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, identification, and purification of murine thymic epithelial cells

胸腺は、Tリンパ球の開発のための重要な器官である。 T細胞のコミットメント、正の選択および負の選択：胸腺ストローマ細胞、胸腺上皮細胞（TECは）は、T細胞発生の複数の段階において特に重要である。しかし、胸腺内のTECの機能は完全には理解されたままになります。記事では、機械的破壊及び酵素消化の組み合わせを使用して、新鮮なマウスの胸腺からTECサブセットを単離するための方法を提供する。方法は、胸腺間質細胞および胸腺細胞を効率的に細胞 - 細胞および細胞 - 細胞外マトリックス接続から解放されると、単一細胞懸濁液を形成することを可能にする。単離された細胞を使用して、マルチパラメータフローサイトメトリーのTECおよび樹状細胞の同定および特徴付けにも適用することができる。のTECは、胸腺では珍しいな細胞集団であるため、私たちも豊かにし、胸腺細胞、胸腺で最も豊富な細胞型を枯渇させることによりTECのを浄化する効果的な方法について説明します。 Folloの細胞生存率の損失はTECの精製の間に最小限に抑えることができるように、翼濃縮を、時間を選別細胞を減少させることができる。精製された細胞は、リアルタイム-PCR、ウェスタンブロット及び遺伝子発現プロファイリングなど、さまざまな下流の分析に適している。プロトコルは、TEC機能の研究と同様にin vitroでのT細胞再構成の開発を推進していきます。

このプロトコルを使用して、胸腺器官成体マウス（セクション2）から除去し、得られた細胞懸濁液を3章で概説されるように胸腺細胞懸濁液を調製した胸腺細胞、造血由来間質細胞および非造血間質細胞から成っていた。 CD45は胸腺細胞とマクロファージや樹状細胞などの造血間質細胞の両方で発現し、造血汎マーカーである。対照的に、TECは、造血起源のものではなく、CD45 15を発現...

Watch this Scientific Journal Video about Isolation, Identification, and Purification of Murine Thymic Epithelial Cells at JoVE.com

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

ここでは、単離、同定、およびマウス胸腺上皮細胞（のTEC）を精製するための効率的な方法を記載する。プロトコルは、通常のT細胞の発生、胸腺機能不全、およびT細胞再構成のための胸腺機能の研究のために利用することができる。

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 ...

isolation-identification-purification-murine-thymic-epithelial

Research

JoVE Journal

Immunology and Infection

31.7K ビュー.  University of Minnesota. ここでは、単離、同定、およびマウス胸腺上皮細胞（のTEC）を精製するための効率的な方法を記載する。プロトコルは、通常のT細胞の発生、胸腺機能不全、およびT細胞再構成のための胸腺機能の研究のために利用することができる。 

ビデオ: Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

M細胞による細菌の取り込みを評価するパイエル板腸管ループアッセイを連結したマウスのアプリケーション

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

免疫・感染学

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

機能と分子生物学的研究のためのプライマリマウスII型肺胞上皮細胞のフローサイトメトリ分離

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

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 ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

ヒト胸腺からの骨髄樹状細胞と上皮細胞の単離

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

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 ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

網膜色素上皮細胞に貪食アッセイのためにブタまたはウシの感光体外側セグメントの大規模精製

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

単離と異なる高齢マウス胸腺移植片の移植。

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

EAK 16-II / EAKIIH6自己組織化ヒドロゲルとFACS精製した胸腺上皮細胞の3-D集約を推進

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.