This work was supported by funding received from the National Institutes of Health (1R01AI083269).

マウスの研究では、ノースカロライナ州チャペルヒル大学の施設内動物管理使用委員会（IACUC）によって承認された、すべての動物のケアは、IACUCガイドラインに従いました。 新生児と成人胸腺の作製<ol><li>ドナーマウスを安楽死する前に、すべての試薬や機器を準備します。 </li><li>オートクレーブ処理または他の適切な方法によって手術器具を滅菌します。すべての外科的処置は、無菌状態を維持し、汚染を避けるために、層流フードの下で実行されなければなりません。ドナーマウスからの胸腺の抽出のために必要なツールを組み立てます。 </li><li>滅菌1×PBS（pH7.4）で60ミリメートル皿を記入し、フードの内側に氷の上に置きます。これは、移植前にドナーマウスから切除簡単ストア胸腺に使用されます。 </li><li>倫理指針に従って、頸椎脱臼に続くCO 2窒息により断頭し、成人ドナーマウスによって新生児ドナーマウスを安楽死させます。 </li><li> Rについて胸腺のemoval：滅菌吸収性ペーパータオルの上に背臥位でマウスを置き、前切開し、70％エタノールでスプレー。 </li><li>皮膚を通して正中切開を行うことで、腹部や胸腔を公開します。胸腔を明らかにするために胸と前肢上の皮膚を折ります。 </li><li>優れた縦隔を公開し、胸腔を前方する振動板と胸郭を介して2つの横方向の切開を行います。胸腺は、二つの白いローブすぐ上に、心臓に隣接するとして表示される必要があります。 </li><li>カプセルを破壊するために、特定のではないながら、微細な鉗子で胸腺を周囲の結合組織を離れていじめます。鉗子で胸郭をバック保持しながら、臓器の下に湾曲した鉗子を配置し、垂直方向に引っ張ることにより、胸腺の2つのローブを抽出するために鉗子の第二の対を使用しています。これは、新生児マウスから胸腺を抽出するための解剖スコープを使用して行うことができます。 </li><li> 60ミリメートルジに胸腺を配置SH結合地峡を介して切断することにより、氷と別々の胸腺葉の滅菌1×PBS（pHは7.4）を含有します。カプセルに損傷を与えない特定すること胸腺葉から任意の破片を除去し、移植のためのセクションの適切な数に胸腺をカット。 </li><li>新生マウスから得た胸腺を操作しないでください。 注：2レシピエントマウス（受信者ごとに1葉）の最大のために使用されます。 </li><li> 4-6レシピエントマウスの最大に成体マウスから得た胸腺を移植。ピンセットを用いて、 図1に示すように、慎重に、大人一人胸腺葉を持ち、手術用はさみを使用して、3つの等しい部分に胸腺をカット。 </li><li>繰り返しは、各ドナーマウスのための1.3〜1.4を繰り返します。胸腺が露出する時間を制限するために、一度に1ドナー胸腺を準備します。 </li></ol> 2.胸腺移植腎臓被膜下<ol><li> 表1に記載されている予め滅菌装置を組み立てます。適切な無菌技術は汚染されたツールや試薬に移植レシピエントを露出しないようにする手順の過程で利用されるべきです。 </li><li>移植前に、各レシピエントマウスの重量を量るとタグ。 </li><li>層流フード中で解剖顕微鏡および麻酔回路を設定します。 </li><li>電気かみそりを使用して、レシピエントマウスの左側を剃ると、髪の毛が切開を作るために使用される領域の周囲に残っていないことを確認します。 </li><li>イソフルラン気化器の電源を入れ、1〜2％の間の用量を用いてマウスを麻酔。前つま先のピンチ次反射の不在を確認することにより、外科的処置を開始する適切な麻酔を確かめます。 </li><li>マウスが適切に麻酔した後、乾燥を防ぐため、次のように移植のためにマウスを準備するために、マウスの目に獣医の軟膏を適用します。 </li><li>剃毛側が上を向いているように右側臥位で解剖顕微鏡下でマウスを置きます。 Starti手術領域の中心にngの、ポビドンヨード（ベタジン）続い使い捨てトランスファーピペット70％エタノールを使用して円を描くように分配します。前切開部を作ることに、エタノール/ベータダイン処理を3回繰り返します。 </li><li>解剖ハサミを使用すると、直接腎臓上記1〜2センチメートルフランク切開を行います。 </li><li>腎臓に隣接する結合組織をつかみ、それが筋肉組織の上に位置するように静かに腎臓を持ち上げるメディア鉗子を使用して。筋肉組織の上に露出し、代わりに腎臓を維持するために、腎臓、腸骨を破壊しないように注意しながら、腎臓下の媒体鉗子の一方のアームを挿入します。 </li><li>腎臓および腎臓被膜の操作が完了するまで、組織の乾燥を防ぐために、各ステップの間、滅菌1×PBS（pH7.4）で腎臓を洗浄。 </li><li>細かい鉗子を使用すると、腎臓からそれを分離するための副腎への最も遠位の腎臓のエッジ付近カプセルをつまんで持ち上げます。 </li><li> 18ゲージを使用してください針は、ドナー胸腺（ 図2A）の準備片を挿入するのに十分な大きさの切開を作ります。時間をかけて移植片の転位を防止するために、可能な限り小さく莢切開を保つために確かめて下さい。 </li><li>カプセルを維持することは離れて腎臓から引っ張っているが、カプセルの下に移植片を挿入し、可能な限り莢切開（ 図2B）から前方まで胸腺をプッシュする微細な鉗子の第二の対を使用しています。 </li><li>腎臓の下から鉗子を外し、静かに切開して元の位置に戻し、腎臓を返します。 </li><li>腹膜壁を縫合し、一度閉じたベータダインを適用することにより、筋肉組織を閉じます。縫合が完了した後、真皮を閉じ、周辺地域にベタジンを適用するために、創傷クリップを適用します。 </li><li>加熱ランプを使用して温めてきたケージにマウスを返します。 </li><li>それは完全な意識とモビリティを取り戻すまで、各術後マウスを監視し、マウスのTHAを置かないでくださいtは他の動物とのケージに次の麻酔を回復していません。 </li><li>必要に応じて術後疼痛管理薬をマウスに扱います。このように1.6mg / mlの濃度で飲料水中のアセトアミノフェンのような術後の鎮痛薬をマウスに与えます。 </li><li>繰り返しは、各レシピエントマウスのための2.3~2.9を繰り返します。 </li><li>マウスは、1時間後に手術内の通常の活動に戻ることを確認してください。移植手順の結果として生じる合併症を決定するために、術後の体重、運動能力、ならびに各マウスの飲料水や給餌活動を監視します。 注：術後の体重減少、術前の体重に比べ、ならびに移動度の変化や飲食に失敗すると合併症を示すことができます。 </li><li>尾ニック経由でマウスを出血、末梢血中のT細胞再構成を監視し、製造業者の仕様に従ってLympholyte細胞分離媒体を使用してリンパ球を分離しました。 T細胞characterizaを達成T細胞マーカーCD3、CD4、およびCD8に特異的な蛍光標識抗体、ならびにそのような蛍光活性化スクシンイミジルエステル8として生き死識別器でリンパ球を染色することによってます。 図3に示すように、分析はシングレット、生細胞およびCD3陽性細胞にゲートを介して達成することができます。 </li></ol>

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The authors do not have any competing financial interests to disclose.

Events regulating thymic selection are poorly understood. Recent studies have demonstrated ontogenic changes in the efficiency of selection. Coupling this approach with different transgenic thymus donor and recipient mice will further facilitate studies to identify the events and parameters that regulate thymic selection. Notably, thymic transplantation is used for the treatment and management of T cell and thymic disorders seen for instance in athymic infants lacking functional T cells and in patients with DiGeorge synd...

胸腺は、T細胞の発達における重要なイベントが1を発生する器官です。レジデント胸腺細胞は、T細胞受容体（TCR）αおよびβ遺伝子の再編成時に、胸腺2の皮質と髄質の地域でlymphostromalと抗原提示細胞（APC）との相互作用のシリーズを受けます。胸腺正の選択は、宿主の主要組織適合遺伝子複合体（MHC）クラスIおよびII分子2-3との関連で抗原性ペプチドを認識する胸腺細胞を産生するために、皮質胸腺上皮細胞（TEC）によって媒介されます。その後の胸腺のネガティブセレクションは、髄TECまたは樹状細胞（DC）、MHCクラスI及びII分子によって結合された3自己タンパク質に由来する本発明のペプチドとの相互作用によって駆動される自己反応性胸腺細胞のパージを伴います。これらのプロセスの最終結果は、広いスペクトルに対応することができ、成熟CD4 +およびCD8 + T細胞のプールの確立であります外来抗原の自己タンパク質4に最小限の反応性を示しながら。 胸腺選択イベントの効率は胸腺成熟、髄質および皮質TEC、胸腺のDCのサブセット組成の頻度、および胸腺前駆体3の源を含む因子の宿主によって影響されます。特に、異常な胸腺選択は、それぞれ、自己免疫5または損なわ正または負の選択から生じる免疫不全の病態をもたらすことができます。胸腺選択を調節する分子のイベントは、しかし、よく理解されていない。 インビトロでは 、このような再凝集胸腺器官培養（RTOC）6のように近づき、胸腺選択に関連する基本的な事象を分析するために有用であるが、完全には進行中のダイナミクスを再現することができないことが証明されています生体内のイベントで 。その結果、この胸腺移植ベースのアプローチは、インビボ 7 でより良い研究の胸腺細胞選択イベントに設立されました</s&gt;アップ。 このプロトコルは、免疫不全SCIDレシピエントマウスに新生児と成人ドナーマウスから胸腺を移植について説明します。この技術は、正および負の選択胸腺、ならびに個体発生中の様々なT細胞サブセットの胸腺の出力を調節する機構の研究を可能にします。最近、このアプローチは、胸腺選択の効率は、初期の自己反応性T細胞の増加した開発、および外来抗原7に特異的な減少T細胞レパートリーをもたらすマウスにおいて生後限定されることを実証するために使用されています。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Decon Laboratories</td>
			<td>2705HC</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>GIBCO</td>
			<td>14190-144</td>
			<td>pH 7.2</td>
		</tr>
		<tr>
			<td>Betadine Solution</td>
			<td>Purdue Products</td>
			<td>67618-150-17</td>
			<td></td>
		</tr>
		<tr>
			<td>18 gauge needle</td>
			<td>BD</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures (1.0 metric)</td>
			<td>Ethicon</td>
			<td>J493G</td>
			<td>18&#34; (45cm)</td>
		</tr>
		<tr>
			<td>AUTOCLIP Wound Clips (9mm)</td>
			<td>Clay Adams&#174; Brand</td>
			<td>427631</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Fisher Scientific</td>
			<td>13-711-20</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr>
			<td>Mouse anti-CD3 Ab</td>
			<td>eBioscience</td>
			<td>11-0031-85</td>
			<td>Clone: 1452C11</td>
		</tr>
		<tr>
			<td>Mouse anti-CD4 Ab</td>
			<td>eBioscience</td>
			<td>48-0042-82</td>
			<td>Clone: RM4-5</td>
		</tr>
		<tr>
			<td>Mouse anti-CD8 Ab</td>
			<td>eBioscience</td>
			<td>25-0081-82</td>
			<td>Clone: 53-6.7</td>
		</tr>
		<tr>
			<td>Lancet</td>
			<td>Medipoint</td>
			<td>Goldenrod 4mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Orange Succinimidyl Ester, Triethylammonium Salt</td>
			<td>Invitrogen</td>
			<td>P30253</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well round botton polypropylene plates</td>
			<td>Corning</td>
			<td>3365</td>
			<td></td>
		</tr>
		<tr>
			<td>1.2mL polypropylene cluster tubes</td>
			<td>Corning</td>
			<td>4401</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL polypropylene round-bottom tubes</td>
			<td>BD</td>
			<td>352002</td>
			<td></td>
		</tr>
		<tr>
			<td>40uM Nylon Cell Strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde Solution, EM Grade</td>
			<td>Electron Microscopy Services</td>
			<td>15710</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Puralube Optical Ointment</td>
			<td>Fisher Scientific</td>
			<td>NC0138063</td>
			<td></td>
		</tr>
		<tr>
			<td>Lympholyte</td>
			<td>Cedarlane</td>
			<td>CL5030</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm cell culture dish</td>
			<td>Corning</td>
			<td>430196</td>
			<td>60mm x 15mm, Sterile</td>
		</tr>
	</tbody>
</table>

isolation and transplantation of different aged murine thymic grafts.

胸腺選択の有効性を制御する機構は不明確なままです。ここで紹介する方法は、自己と外来抗原に特異的なT細胞の開発および選択の分析、in vivoで可能にします。アプローチは、免疫不全SCIDレシピエントに様々な高齢のマウス由来の胸腺移植片の移植を必要とします。比較的短い期間にわたって受信者は、完全に移植胸腺移植片由来T細胞で再構成されています。唯一の胸腺細胞の分離時に胸腺を播種すると、選択を受け、成熟T細胞へと発展します。このように、年齢依存性胸腺イベントの関数として移植T細胞の性質および特異性の変化を評価することができます。技術的専門知識が成功した胸腺移植のために必要とされていますが、この方法は、 生体内に起因する病態の広い範囲または異常な胸腺機能および/ ​​またはhomeostasの結果を研究するためのユニークな戦略を提供しますです。

この方法の成功は、最小限の外科手術による外傷ならびに腎臓被膜下に移植片の正確な位置決めに依存しています。胸腺移植片は、図1に示すように、その後の移植のために適切なサイズの胸腺のセクションを確実にするために切断されるべきである。 図1の模式に続いて、胸腺は、新生児または成体マウスから一貫して再現性のT細胞移植の結果と成功被膜下移植の?...

Watch this Scientific Journal Video about Isolation and Transplantation of Different Aged Murine Thymic Grafts. at JoVE.com

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

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 ...

isolation-and-transplantation-of-different-aged-murine-thymic-grafts

Research

JoVE Journal

Immunology and Infection

13.7K ビュー.  University of North Carolina at Chapel Hill. 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. 

ビデオ: Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

B細胞のCD40 -活性化のモデルマウス

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

免疫・感染学

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

のRNAの単離緑膿菌マウス消化管に定着

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

フローサイトメトリーによる胸腺正と負の選択の検討

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

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, 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 ...

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 ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

単離とマウス骨髄由来の単球の静脈注射

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

マウスの小腸リンパ球の単離およびフローサイトメトリーキャラクタリゼーション

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

調製及び器官胸腺スライス培養の応用

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

単離とマウスリンパ球の活性化

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...