この研究は、厚生労働省の難病研究に対する厚生医療大学の助成金や厚生労働科学研究助成金によって支援されました。

実験議定書は、国防医科大学動物研究委員会(16058年)によって承認された。動物は標準的な実験室のチャウ(クレア日本株式会社、東京、日本)で維持された。実験動物は国立衛生研究所のガイドラインに従って治療された。
1. リンパ球の採取と分離
注:リンパ球は新鮮で保存できないため、実験ごとにラットから採取する必要があります。さらに、腸管-熱帯リンパ球は、それらが循環する胸部管から直接収集されなければならない。すべての手順は6時間以内に完了する予定で、すべての器具、手袋、表面はきれいです。動物を生理的に安定した状態に保つためには、実験で使用されるすべての生理食塩水および他の液体を暖かく保つべきである。
<ol><li>ヘパリン曲線(半径約5mm)のように、高温(約80~90°C)に浸漬した後、缶詰チューブ(直径1mm)を形成し、数時間前から粘着テープを使用してプラスチック製の板に固定します。</li>
	<li>ミダゾラム(2mg/kg)、ドミトール(0.15mg/kg)、ベトゥルファール(2.5mg/kg)の混合物を腹腔内注射で雄のウィスターラット(8-12週間)に麻酔し、ヘアクリッパーを使用して腹部の体毛を切断します。髪が腹腔に入らないように剃った後、しっかりと髪を取り除きます。</li>
	<li>つま先ピンチにより、手術に必要な安定した麻酔の深さが得られていることを確認した後、中線から左肋後部領域に水平に外科用ハサミで切開を行う。光の下で血管を見て頭頂腹の血管を損傷しないように注意してください。</li>
	<li>ぬれたガーゼで胃を包み、胃を体の右外側にやさしく動かして後腹膜器官を明らかにする。外科的はさみを使用して脊椎の勃起筋と脂肪組織の間にノッチを挿入し、指でぶっきらぼうに分離する。</li>
	<li>胸部管の周りの結合組織を慎重に取り除きます。過剰な結合組織は年齢とともに増加する。胸部管の長さが20mmになるまで、横隔膜の左の十字架のすぐ下から胸管を露出させます。胸部ダクトと大タオルの間の密着性を、精密なピンセットや湿った綿棒でそっと分離します。<ol>
<li>一部のラットは、リンパ叢(クモの巣のように広がる小さな胸部管)の高い位置のために、胸部管または短い胸管を横切る腹部動脈から分岐する動脈を持っているので、このプロセスを慎重に実行します。浮腫を誘発しないように、胸部管への不必要な接触を避けてください。</li>
	</ol>
</li>
	<li>横隔膜の左の十字架のすぐ下の胸管に弦(3-0シルク)で合字を適用します。尾柱胸管が歪んでしまいます。</li>
	<li>手術用ハサミの刃先を刺して腹壁に穴(直径5mm)を作り、ヘアピン湾曲したカヌルチューブを通過させ、一点で腸筋にチューブをリゲートします。10 U/mLヘパリンを含む通常の生理液で缶詰チューブを充填します。</li>
	<li>歪んだ胸部管の下にひも(3-0シルク)を置いた後、缶詰チューブの鋭く切断された端で胸部管を刺し、尾部に向かって約5mmに向かってカニューレートし、胸部管を固定用の缶詰管でリゲートします。</li>
	<li>脱水を防ぐために生理食塩水を供給するには、精密ピンセットを使用して胃のアントラムの前壁に穴(直径3mm)を作成し、シリコンチューブ(直径2mm)を幽門リングを通して十二指腸に渡します。傷を縫い上げ、ボルマンのケージで動物を維持した後、3 mL/hの流量でシリコンチューブから各ラットの十二指腸に糖を含んだ生理食動物を注入し始める。ケージ全体をペーパータオルで覆い、暖かく保ちます。</li>
	<li>6 U/mLヘパリン、10%のウシ胎児血清、RPMI 1640培地(pH 7.4;材料表を参照)を含む氷上の50mL円錐形チューブの蓋の中央の穴にcannulationチューブを固定し、氷の上に胸部管リンパ球(TDl)を収集します。チューブ内部のフィブリン血塊形成によるカニューレート管閉塞を防止するために、チューブの先端を円錐管の壁に接触しないように注意してください。約1.0 x 10 8〜109リンパ球を含むリンパ液(約20mL)は、6時間で得ることができる。完全麻酔下でリンパ球を得るために、麻酔の面が監視され、リンパ流が適切に観察される。同じ麻酔薬は、ラットがボールマンのケージに入れられた約3時間後に同じ用量で加え、6時間連続的に深部麻酔を得る。</li>
</ol>2. カルボキシフルオレセインジアセテート・スハクニミジルエステル(CFDSE)のリンパ球標識
<ol><li>ジメチルスルホキシド(DMSO)でCFDSE(材料表)を15.6mM(500 μgのCFDSEをDMSOの60 μLに溶解)に溶解します。</li>
	<li>インキュベートリンパ球(1 x 108〜109)をRPMI 1640の50mLで、CFDSE溶液50μLを37°Cで30分間用いた10を前述した。</li>
</ol>3. 微小血管研究の実験
<ol><li>ミダゾラム(2mg/kg)、ドミトール(0.15mg/kg)、ベトゥルファール(2.5mg/kg)の混合物を腹腔内注射でレシピエントラット(8-12週間)に麻酔し、つま先ピンチで麻酔の深さを確認します。毛髪の汚れを最小限に抑えるために、毛皮を手入れする前に腹部を濡らします。次に、正中線切開を介してレシピエントウィスターラットの腹部を開く。</li>
	<li>ガラススライド(24 x 50 mm)で覆われた中央の周りに長方形の穴を持つポータブルステンレス鋼板(約120 x 300ミリメートル)にラットを置きます。観察のためにPを含む、イレラルセグメントの約10cmを選択してください。</li>
	<li>腸をできるだけ暖かく保ち、リン酸緩衝生理食塩分(PBS)を37°Cに温めて湿ら。 PBSで小腸を覆うために使用されるガーゼを浸します。</li>
	<li>2%のイソフルラン(材料表を参照)で連続麻酔を与えながら、顕微鏡のステージにスライドを置き、いくつかのHEVがセローザを通って走っているPPの微小循環の観察に適した領域を選択します。P のサイズの範囲;より大きな物はCLSMを用いた微小循環の観察に適している。小腸は場所でまっすぐではありません。長さ2cm以上の直線セグメントは、張力なしで観察に適しています。</li>
	<li>隣接する腸管セグメントと腸間腸をPBSで浸した吸収性綿で覆います。2つのロールコットンボールの間に腸セグメントを配置し、ラットの心拍と呼吸によって振動するのを防ぐために、ラットの体から可能な限り遠くに配置します。</li>
	<li>1 mL シリンジを使用して、CFSE 標識 TDL (1 x 108 細胞) をレシピエントラットの頸静脈にゆっくりと注入します。急速な静脈注射は、全身循環に影響を与える可能性があります。</li>
</ol>4. リンパ球の微小循環
<ol><li>CLSMを使用してPのマイクロバスキュアチュアのTDLを継続的に監視し、30 s間隔でタイムラプス写真を使用して3時間コンピュータに記録します。セローサからPPsのHEVまでの深さは約25μmで、30μmの深さまでストロマと毛細血管を観察することができます。</li>
	<li>テキサスレッドデキストラン(25mg/kg)を各レシピエントラットの頸静脈に注入して血流を染色し、Hoechest 33342(5mg/kg)を注入して細胞核を染色します。</li>
	<li>(オプション)リンパ球のダイナミクスを定量化するために、HEVに30秒以上付着するリンパ球を「接着性リンパ球」と定義し、HEVからストロマに移動するリンパ球を「リンパ球の移行」と定義する。次に、移動の平均割合(リンパ球/粘着リンパ球+移動リンパ球)を視野(約0.3mm2)で計算する。</li>
</ol>

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著者らは開示するものは何もない。

ここでは、ナイーブな腸管熱帯リンパ球を収集し、ラットPでの移動を観察するためのプロトコルについて説明しました。これらの手順は、リンパ球がPの微小血管構造でどのように動くかを明らかにし、正常または薬用状態下でそれらのダイナミクスを視覚的に比較することを可能にする。これらのダイナミクスの直接観察は、観察期間は数時間に制限されているが、いくつかの薬物による?...

ペイアーのパッチ(P)は、小腸の層状層の数百のリンパ卵胞で構成されています。PPは、卵胞、卵胞間領域、および卵胞の下部に位置する胚葉中心に分けられ、そこでリンパ球は抗原提示によって刺激される。不十分なリンパ管はなく、抗原は上皮細胞層を介して腸管腔から層状のプロプリアに侵入する。リンパ様卵胞を覆う上皮領域は、毛包関連上皮と呼ばれ、その中に特殊な散在するM細胞が粘膜抗原を取り込む。M細胞は、その後、高等内皮細胞(HEV)の内皮を介してPに流入する素朴なリンパ球に向かって提示される、発光側および抗原からの抗原を取り込む。PPは腸内免疫に重要な役割を果たし、炎症の初期段階に関連しています。多くの分子相互作用は、接着分子、ケモカイン2、3、およびスフィンゴシン-1リン酸4を含む、二次リンパ系器官(SLA)へのリンパ球の入り口を含む。したがって、多くの期待される治療標的がある。したがって、PP内のリンパ球ダイナミクスを観察することで、炎症の非常に初期の段階を垣間見ることができ、いくつかの有望な薬物の有用性を調べることができます。
この方法は、いくつかの手順(胸管5 へのカヌリン化および採取されたリンパ球への注射後のリンパ球および長期観察)を含む、PPにおけるリンパ球の移行に焦点を当てています。これらの手順は複雑で、これまでのレポートで各手順がどのように実行されたかを正確に確認することは困難であったため、ここで、観察を成功させるためのヒントをいくつか挙げた。例えば、胸部管への管のカヌレーションは非常に困難であり、そして、カヌリン化の最初の成功率は50%未満であった。しかし、我々は方法を改善し、80%を超える成功率を達成しました。我々は、いくつかの条件下でリンパ球の経経皮移動の定量的評価を可能にするために成功した観察に必要な他のいくつかのヒントをこの原稿で述べた。
これまでの報告では、インドのインクを静脈内注入してPP6の血管構造を染色する、または顕微鏡が単焦点7であるなど、時間の経過に伴う3次元的変化を理解することは困難であった。近年、Kaedeマウスのようないくつかの光変換可能な蛍光タンパク質トランスジェニック動物を用いた観察法が、生体内における系統的な細胞の動きを明らかにしている。もう一つの研究は、PPs9からのリンパ球出口のCD69独立したシャットダウンを明らかにした。分析能力が高いため、共焦点レーザー走査顕微鏡(CLSM)を使用しました。今では、高解像度の画像を簡単に取得し、リンパ球ダイナミクスを分析するためにそれらを使用することができます。
本報告では、PPにおけるリンパ球移動を評価するための一連の方法を示した。まず、リンパ球を採取する胸部管缶取の精製方法を示した。第2に、顕微鏡観察の下で可能な限り目的の器官を維持するために、いくつかの方法で観察方法を改善し、3時間の高品質な画像を得ることを可能にしました。第三に、リンパ球移動の細胞の動きを定量化し、いくつかの薬の効果を評価した。これらの改変されたプロトコルは、粘膜免疫学評価の開発に寄与する。

<table><tbody><tr><td>A1R+</td><td>Nikon</td><td></td><td>Comfocal Laser Scanning Microscopy</td></tr><tr><td>Carboxyfluorescein diacetate succinimidyl ester</td><td>Thermo Fisher Scientific</td><td>C1157</td><td></td></tr><tr><td>Hoechest 33342</td><td>Thermo Fisher Scientific</td><td>H3570</td><td></td></tr><tr><td>Isoflurane</td><td>Wako Pure Chemical Industries</td><td>099-06571</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>GIBCO</td><td>11875093</td><td></td></tr><tr><td>Texas Red&amp;ndash;dextran</td><td>Thermo Fisher Scientific</td><td>D1863</td><td></td></tr></tbody></table>

microscopic observation of lymphocyte dynamics in rat peyer's patches

ナイーブリンパ球は、生理学的状態下で血液からリンパ組織に再循環し、腸免疫における重要な現象として一般的に認識されています。ペイヤーのパッチ(P)や腸間膜リンパ節などの二次リンパ器官の間質は、ナイーブリンパ球が抗原を感知する場所である。ナイーブリンパ球は、高い内皮小静脈、PPへの参入のポータルに到達するために血流を循環します。一部の免疫調節剤はリンパ球の移動に影響を与えると推定されるが、微小循環ダイナミクスの正確な評価は非常に困難であり、生体内でリンパ球の移動を観察する方法を確立することは、正確なメカニズムの解明に寄与することができる。リンパ管からリンパ球を採取し、ラットPの腸管-熱帯リンパ球の詳細なダイナミクスを観察する方法を改良した。生体内でラットPを観察する共焦点レーザー走査顕微鏡を選び、タイムラプス写真を用いて記録しました。リンパ球動態の解析に貢献できる鮮明な画像を得ることができるようになりました。

リンパ球からのリンパ球の採取 胸部管のカヌレーションのためにラットを準備するには、 図1 に示すように緊張した胸管に切開し、次に図 2に示すようにボルマンのケージにラットを維持する。
リンパ球が十分に採取されると、約107〜108/mLリンパ球を含む約20mL/6hのリンパ液を得ることができる。</su...

Watch this Scientific Journal Video about Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches at JoVE.com

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

ここでは、経胸管リンパ球を採取し、ラット・ペイアーのパッチ中の腸管リンパ球の移動をタイムラプス写真を用いて3時間観察するための正確な方法について述べた。この技術は、炎症性条件下でリンパ球の動態がどのように影響を受けるかを明らかにすることができる。

ラット・ペイアーのパッチにおけるリンパ球ダイナミクスの顕微鏡観察

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important ...

microscopic-observation-of-lymphocyte-dynamics-in-rat-peyer-s-patches

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

3.9K ビュー.  National Defense Medical College. ここでは、経胸管リンパ球を採取し、ラット・ペイアーのパッチ中の腸管リンパ球の移動をタイムラプス写真を用いて3時間観察するための正確な方法について述べた。この技術は、炎症性条件下でリンパ球の動態がどのように影響を受けるかを明らかにすることができる。

ビデオ: Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

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&#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

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer&#39;s patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes.
Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on&#160;TFH and GC B cell subsets.

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer&#39;s Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

体液性免疫のキープレーヤーの解明: 高度なマウス Peyer のパッチからのリンパ球の隔離のプロトコルを最適化

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense ...

がん研究

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 ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

マウスのパイエル板のリンパ球や樹状細胞を単離し、免疫染色

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

胚中心の高解像度生体内ストライプ照明顕微鏡

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

イメージング免疫学的シナプスダイナミクス内皮平面内細胞モデル

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

マウス小腸管免疫系からリンパ球を分離すること

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Traction Force Microscopy to Study B Lymphocyte Activation

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements from an experimentally observed displacement field produced by a cell pulling on an elastic substrate. Here, we adapted TFM to investigate the spatial and temporal structure of the force field exerted by B cells when activated by antigen engagement of the B cell receptor. Gel rigidity, bead density, and protein functionalization must be optimized for the study of relatively small cells (~ 6 &#181;m) that interact with, and respond specifically to ligands for cell surface receptors.

Here we present a protocol used to perform traction force microscopy experiments on B cells. We describe the preparation of soft polyacrylamide gels and their functionalization, as well as data acquisition at the microscope and a summary of data analysis.

Bリンパ球活性化を研究するための牽引力顕微鏡

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements ...

生物工学

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

マウスの皮膚腫瘍からリンパ管を介して白血球出口の定量化

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...

生物学

Isolation of Lymphocytes from a Mouse Peyer&#39;s Patch

Source: Yazicioglu, Y. F., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/58490">Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer&#39;s Patches.</a> J. Vis. Exp. (2018)
In this video, we demonstrate the isolation of lymphocytes from a harvested mouse Peyer&#39;s patch. The isolated lymphocytes can be used for further analysis.

マウスパイエルパッチからのリンパ球の単離

Source: Yazicioglu, Y. F., et al. Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer&#39;s ...