このプロジェクトは、健康補助HL087088（MK）とHL18208（MK）の国立研究所によってサポートされていました。

1。ヒトTリンパ球の分離<ol><li>健康なドナーからのヒトの血液を入手してください。血液は、次のステップに進む前に室温（〜30分）に冷却してください。 </li><li> 8 mLの丸底ポリスチレンチューブに室温多形密度勾配媒体のピペット3mLの。ゆっくりと多形のメディアの上に全血3 mLを加える。 2種類の試薬の混合を避けることが重要です。 </li><li>室温で45分間、500 × gでチューブを遠心する。 </li><li>遠心分離に続いて、末梢血単核細胞（PBMC）は一番上のセルの層に他の血液成分から分離している。 PBMC層は、最初の曇りバンドとして、トップダウンから、表示されます。 </li><li>慎重にPBMC層上に血の透明な黄色の上相を、削除し、15 mlまたは50 mlコニカルチューブにPBMC層を転送するためにP1000マイクロピペットを使用してください。 </li><li> 5分ごとに時間を500 × gで細胞を遠心分離し、PBSで2回PBMCを洗ってください。上清は、各洗浄後やや曇っ​​たようになります。 </li></ol> 2。ヒトTリンパ球の培養<ol><li>ピペットを用いて、10％FBS、1％ペニシリン/ストレプトマイシン、及び1μg/ mlのフィトヘムアグルチニン（PHA）を含む20mLのRPMI 1640培地でT - 75培養フラスコにPBMCを転送する。 </li><li> 37 ° C、5％は少なくとも1時間、および最大24時間CO 2。このステップでは、フラスコの表面に付着なる単球は、懸濁液に残っているリンパ球から分離することができます。短いインキュベーション（1時間）は、このステップで使用されている場合は、手順2.1で指定されているPHAで補うことなく、10％FBSおよび1％ペニシリン/ストレプトマイシンを含むRPMI 1640培地を使用することは可能です。 </li><li>慎重に、フラスコからメディアのすべてを削除する50 mLコニカルチューブにそれを追加し、5分間500 × gで遠心。 </li><li>現在は主にリンパ球を含む細胞ペレットを、再懸濁し、そして10％FBS、1％ペニシリン/ストレプトマイシン、及び1μg/ mlのPHAを含む25mLのRPMI 1640培地を含む新たなT - 75フラスコに細胞を移す。 </li><li> 3日間（PBMCの最初のインキュベーションが一夜だった場合2日間）を37℃でインキュベートする。成長の24時間後、それは新鮮な培地の15〜20 mLを加え、より大きなT - 175フラスコに転送する必要があるかもしれません。 </li><li> 3日後、フラスコ、50 mLコニカルチューブに移すからメディアおよび一時停止中のリンパ球を除去するためにピペットを使用してください。 5分間、500 × gで遠心する。 </li><li>新しいT - 75または25mLの（T - 75）または50 ml（T - 175）10％FBS、1％ペニシリン/ストレプトマイシン、および20 ngのRPMI 1640を含むT - 175フラスコに細胞ペレットおよび転送細胞を再懸濁します/ mLのヒトIL - 2またはIL - 15。 </li><li> 4-7日のためにリンパ球が成長する。 T - 75フラスコで始まる場合、文化は1〜2日後に拡大し、T - 175フラスコに転送する必要があるでしょう。 </li></ol> （3） 体外リンパ球遊走アッセイで <ol><li>遊走アッセイの前に1日、コートで20μg/ mLのタンパク質一晩4 PBSでまたはGとグラスボトム0.17ミリメートルディッシュ℃の</li><li> PBSで皿を洗う。 </li><li>ディッシュにPBS溶液にこれらの試薬を加え、室温で4時間インキュベートすることにより、人間のICAM-1/Fc（10μg/ mLの）と、ヒトSDF - 1（2μg/ ml）を固定化する。 </li><li>第一血球計を使用してT - 175フラスコで培養中の細胞密度を決定することによって、Tリンパ球を準備します。約2〜5 × 10 5細胞遊走の分析のために適切な細胞密度を達成するために皿ごとに使用する必要があります。 </li><li> PBSで2回、Tリンパ球を洗浄し1 mg / mLのD -グルコースを含む1 mLのL - 15培地に再懸濁します。 </li><li>広範囲にPBSでICAM-1/FcとSDF - 1を塗布した皿を洗う。 </li><li>皿に1 mLのメディアにおけるTリンパ球を移し、37でそれらをメンテナンス℃に</li></ol> 4。 NISの要素ソフトウェアを用いた動画像のキャプチャ<ol><li> NISの要素のソフトウェアを開きます。 </li><li>カメラの設定メニューでは、ライブイメージングと画像キャプチャの両方のモードとして、&quot;2x2ビニング&quot;を選択してください。 </li><li>アプリケーションメニューに移動し、実行定義/実験を選択してください。 </li><li>画像と画像のキャプチャシーケンスの長さの合計との間の時間の長さを選択してください。 </li><li>画像の集録を開始するには&quot;Run&quot;ボタンを押してください。 </li><li>細胞を追跡し、移行パラメータ（速度、パスの長さ、変位等）ImageJを、AutoQuantまたはVolocity（図1）を含むいくつかのソフトウェアパッケージ、のいずれかを使用して定量することができる。 </li><li>各セルとタイムポイントを収集する（図2）と、原点での各セルに共通の出発点（図3）をもつグラフ上に投影されるために生成するには&quot;クモの巣のプロットを、&quot;XY座標。 </li></ol> 5。代表的な結果 IL - 2またはIL - 15、Tリンパ球のいずれかの存在下での培養の6日目として正のCD3の染色だけでなく、肯定的なCD4および/またはCD8染色によって決定される細胞の&gt; 98％を、占める。 IL - 2の場合、我々は83％のCD4 +細胞を、15％CD8 +と&lt;1％のCD4 + CD8 +細胞を発見した。 IL - 15の場合、我々は88％のCD4 +細胞を、11％CD8 +と&lt;1％のCD4 + CD8 +細胞を発見した。 ICAM-1/SDF-1基板上でのTリンパ球の移行時に、細胞は1時間の期間にわたって持続することができる約15μm​​/分の速度を示した。抗LFA - 1リガンド阻害抗体は、深刻な移行を阻害するとして。ICAM-1/SDF-1でTリンパ球遊走には、LFA - 1媒介接着に依存しています<img src="/files/ftp_upload/2017/2017fig1.jpg" alt="図1" /> 図1。 Tリンパ球の移行の細胞追跡。Tリンパ球全血から分離し、6日間IL - 2またはIL - 15の存在下で培養を10μg/ mLのICAM - 1を塗布したガラス底皿に付着して移行させたと濃度2μg/ mLのSDF - 1。画像は、30分間10秒ごとに撮像した。細胞は、それぞれの画像で同定し、Volocityソフトウェアを使用して時間をかけて追跡した。この映画は、〜15μm/分の速度でTリンパ球のランダムな移行を示しています。 <a href="http://www.jove.com/files/ftp_upload/2017/2017_Kim_Movie1.mov">ビデオを見るにはここをクリック。</a> Tリンパ球の移行の動画1。細胞のトラッキング。全血と6日間IL - 2またはIL - 15の存在下で培養から分離されたTリンパ球は、10μg/ mLのICAM - 1および2μg/ mLのSDF - 1を塗布したガラス底皿に付着して移行させた。画像は、30分間10秒ごとに撮像した。細胞は、それぞれの画像で同定し、Volocityソフトウェアを使用して時間をかけて追跡した。この映画は、〜15μm/分の速度でTリンパ球のランダムな移行を示しています。 <img src="/files/ftp_upload/2017/2017fig2.jpg" alt="図2" /> 図2時空間セルの位置。各セルのXY座標がVolocityソフトウェアを使用して、各時点が得られた。 <img src="/files/ftp_upload/2017/2017fig3.jpg" alt="図3" /> 図3。&quot;スパイダーウェブのプロット。&quot;各セルのXYTの座標を使用して、15セルがランダムに選択し、原点を共通の出発点でプロットした。クモの巣プロットを比較すると、異なる実験条件間移行の違いを簡単に視覚的描写を与える。制御条件の下でTリンパ球遊走（左パネル）および抗LFA - 1リガンド阻害抗体（右パネル）の存在下でプロット。

<ol>
	<li>Hyun, Y. M., Chung, H. L., McGrath, J. L., Waugh, R. E., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+integrin+VLA-4+localizes+to+the+lamellipodia+and+mediates+t+cell+migration+on+VCAM-1.">Activated integrin VLA-4 localizes to the lamellipodia and mediates t cell migration on VCAM-1.</a> J Immunol. 183, 359-369 (2009).</li><li>Morin, N. A., Oakes, P. W., Hyun, Y. M., Lee, D., Chin, Y. E., King, M. R., Springer, T. A., Shimaoka, M., Tang, J. X., Reichner, J. S., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonmuscle+myosin+heavy+chain+IIA+mediates+integrin+LFA-1+de-adhesion+during+T+lymphocyte+migration.">Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration.</a> The Journal of experimental medicine. 205, 195-205 (2008).</li><li>Hyun, Y. M., Lefort, C. T., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+integrins+and+their+ligand+interactions.">Leukocyte integrins and their ligand interactions.</a> Immunologic research. , (2009).</li></ol>

利害の衝突は宣言されません。

この実験では、一次ヒトTリンパ球の運動性を分析するためにシンプルなシステムについて詳しく説明します。vitroでの遊走のアッセイでは多くの分子と種々の細胞型の運動に関与するシグナル伝達経路の役割を解明するために使用されている。我々のプロトコルから独自の実験を設計するとき心に留めておくべきいくつかの重要なコントロールが含まれます：1）、ウシ血清アル...

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<td>RPMI 1640</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11875</td>
<td>&nbsp;</td>
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<td>FBS</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH300070.03</td>
<td>&nbsp;</td>
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<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
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<td>1-Step Polymorphs</td>
<td>&nbsp;</td>
<td>Accurate Chemical</td>
<td>AN221725</td>
<td>&nbsp;</td>
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<td>PHA</td>
<td>&nbsp;</td>
<td>Remel/Fisher Scientific</td>
<td>R30852801</td>
<td>&nbsp;</td>
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<td>Rec. human IL-2</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
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<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>247-IL</td>
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<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>19459</td>
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<td>Rec. human ICAM-1/Fc</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>720-IC</td>
<td>&nbsp;</td>
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<td>Rec. human SDF-1</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
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human t lymphocyte isolation, culture and analysis of migration in vitro

Tリンパ球の遊走は、他の細胞や細胞外マトリックスタンパク質と発現するリガンドと細胞表面のインテグリンの接着剤の相互作用を伴います。低親和性状態からセル最先端で高親和性状態へのインテグリンの正確な時空間的活性化は、Tリンパ球遊走に重要です。<sup&gt; 1</sup&gt;。同様に、セルの後縁の撤回は、または腹肢、永続的なインテグリン依存性のTリンパ球の運動性を維持する上で必要なステップです。<sup&gt; 2</sup&gt;。白血球の過度の募集と遊走を阻害する手段として、自己免疫や炎症性疾患のターゲットのインテグリンへの多くの治療的アプローチ<sup&gt; 3</sup&gt;。ヒトTリンパ球遊走を調節する分子事象を研究するために、我々が利用してきた<em&gt; in vitroで</emTリンパ球が血管から募集中遭遇する環境を模倣した二次元の基板上に細胞の移動を分析する&gt;システム。 Tリンパ球は、最初の人間のドナーから単離され、その後刺激し、7〜10日間培養されています。アッセイ中に、Tリンパ球は、細胞間接着分子-1（ICAM - 1）、インテグリンLFA - 1のリガンド、および間質細胞由来因子-1（SDF - 1）でコーティングされた基板上に付着して移行することが許可されています。我々のデータは、Tリンパ球が〜15μm/分の回遊速度を示すことを示している。 Tリンパ球遊走は、インテグリンの阻害によって抑制することができる<sup&gt; 1</sup&gt;または細胞移動を調節する細胞性アクトミオシンの機械の阻害剤による<sup&gt; 2</sup&gt;。

Watch this Scientific Journal Video about Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro at JoVE.com

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

Tリンパ球遊走には、リンパ器官、血管系から出口へのホーミング中に発生し、末梢組織に入る。ここで、我々は、Tリンパ球の遊走を分析するために使用できるプロトコルを記述する<em&gt; in vitroで</em&gt;。

移行のヒトTリンパ球の単離、培養および解析インビトロ</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

human-t-lymphocyte-isolation-culture-analysis-migration

Research

JoVE Journal

Immunology and Infection

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

46.1K ビュー.  University of Rochester. Tリンパ球遊走には、リンパ器官、血管系から出口へのホーミング中に発生し、末梢組織に入る。ここで、我々は、Tリンパ球の遊走を分析するために使用できるプロトコルを記述する in vitroで。

ビデオ: Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

プライマリーヒトT細胞のpiggyBacトランスポゾンシステムの変更

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

免疫・感染学

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

表現型分析のためのヒト皮膚からリンパ球単離と<em&gt; ex vivoで</em&gt;細胞培養

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

マウスナイーブCD4<sup&gt; +</sup&gt; T細胞の単離と<em&gt;インビトロ</em&gt; T細胞サブセットへの分化

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

炎症真皮における撮像CD4 T細胞間質への移行

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

ヒト全血中のナチュラルキラー細胞溶解活性のフローサイトメトリー分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

ヒト子宮頸管ガンマ・デルタT細胞の単離およびフローサイトメトリー分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用してリンパ球血管外漏出の分析<em&gt;インビトロ</em&gt;ヒト血液脳関門のモデル

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

ひと単球走化性の in VitroおよびIn Vivoマウス リンパ球の定量化

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

離とマウスとヒトの肺胞マクロファージの体外培養

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

生体外でマウス縁帯 B 細胞のせん断流動誘起移行の解析

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...