著者らは、E1E4抗体の種類のギフト用サリー·ロバーツ（バーミンガム大学、バーミンガム、英国）に感謝したいと思います。この作品は、国立がん研究所（4R00CA137160-03）からの助成金によってサポートされていました。

1。カドヘリンの文化のための準備<ol><li>金属製のいかだグリッドは、多くの第一分化過程を妨げる可能性のある残留物を除去するためにクロム硫酸で処理することができます。一時間のために硫酸を含有するガラスビーカーに浸した金属グリッドは、その後継続的に水道水で一晩洗浄した。すすぎ一晩後、いかだグリッドは、蒸留水で3-5時間のためにリンスする必要があります。 </li><li>ラフトのサポートを提供するために、互いに等しい距離で約0.5cmいかだの3辺を曲げる。金属グリッドはその後オートクレーブする必要があります。 </li><li> 2.2グラム炭酸水素ナトリウムと4.8グラムのHepesを追加することによって、10X再構成バッファを準備します。 0.05 MのNaOH 100mlに溶解する。濾過滅菌し、-20℃で5ミリリットルのアリコートに格納</li><li>重炭酸ナトリウムなく、10X DMEMを準備します。濾過滅菌し、-20℃で5ミリリットルのアリコートに格納</li><li>マウス上皮細胞増殖因子を準備します。EGF 100mgのAとBの10 mgを溶かし、10ミリリットルのSAは、それぞれのddH 2 0脱。 EGFおよびBSAを結合し、100ミリリットルの合計体積の脱イオンのddH 2 0の80ミリリットルに追加します。ろ過滅菌し、アリコート（5ml）中、-20℃に保管しE培地の各リットルに1 mg / mlのEGF（最終濃度5 ng / ml）を5 mlを加える。 </li></ol> 2。コラーゲンゲルの調製<ol><li> Oneコラーゲンゲルは、それぞれのいかだに必要とされています。コラーゲンは凝固を防止するために氷上で保存する必要があります。コールドラット尾コラーゲンI型（最終濃度3から4 mg / ml）を、0.3ミリリットル10X再構成バッファ、0.3ミリリットル10X DMEM AND1-2×10 6マウスの2.4ミリリットル：各コラーゲンゲルは、以下から構成されるコラーゲンミックスの3ミリリットルを必要とするフィーダー細胞として3T3線維芽細胞のJ2。ラット尾コラーゲンの高濃度溶液の場合は、正しい仕事濃度を達成するために、0.02 M酢酸で希釈します。 </li><li>あなたが必要としてコラーゲンの量のマスターミックスを計算します。コラーゲンゲルの数（ラフト）、10X reconstitを決定utionバッファ、10X DMEMとJ2線維芽細胞が必要です。コラーゲンの粘度のために3月4日余分なゲルの各マスターミックスに十分含まれています。 </li><li>コラーゲンゲルを準備するには、J2線維芽細胞をトリプシン処理し、培地で中和する。 50 mlコニカルバイアル内のすべての線維芽細胞と場所を兼ね備えています。行うことができますラフトの数を決定するために細胞をカウントします。再び、1×10 6線維芽細胞がいかだあたりしたがって、コラーゲンゲルごとに必要とされています。低速度で細胞をスピンダウンする。 </li><li>計算されたラフトの数の線維芽細胞ペレットを再懸濁し10X再構成バッファの適切な量で、10X DMEMとコラーゲンから培地を吸引除去する。早すぎると凝固からゲルを防ぐために、最後のコラーゲンを追加します。コラーゲンが追加されると、すぐに混ぜますが、静かにゲル化気泡の導入を防ぐことができます。ゲルは、適切なpHの示す赤みがかったオレンジ色でなければなりません。ゲル混合物があまりにも黄色である場合、フィルタ滅菌1Nの滴のカップルを追加するNaOHおよび正しい色を得るために静かに混ぜる。 </li><li>コラーゲンの3ミリリットルを追加します。線維芽細胞の混合物を6ウェル培養皿のウェルに。コラーゲン混合物は、泡の生成を回避するために各ウェルの側をゆっくりとピペッティングする必要があります。 37℃の組織培養インキュベーター内でプレートを置き、30分間固化することができます。 </li><li> 30分後、固化したコラーゲンゲルの上にEGFをE培地3 mlを追加し、インキュベーターにプレートを返します。ゲルは、少なくとも1日に37°Cに保持しますが、4日以内に使用する必要があります。最適な結果を得るには、インキュベーションの2日後に発生します。 </li></ol> 3。差別化のために準備ケラチノサイト<ol><li>低い通路ケラチノサイトは約70％コンフルエントに成長する必要があります。それぞれのいかだには、1×10 6ケラチノサイトが必要になります。ケラチノサイトをトリプシン処理およびEGFのE培地で中和する。行うことができますラフトの数を決定するために細胞をカウントします。低SPEで細胞をスピンダウンED。計算されたそれぞれのラフト文化のEGFとE培地1ml中にペレットを再懸濁します。 </li><li>傾斜6ウェルプレートによるコラーゲンゲルから培地を吸引除去する。各ゲルにEGFと新鮮なE培地2 mlを追加します。それぞれのゲルには、慎重にも内にすでに存在するE培地2mlに再懸濁したケラチノサイトの1ミリリットルを追加します。ゆっくりピペットケラチノサイトに加えて、メディアの2 mlを、ゲルの上に秋の滴下戻ってみましょう。プレートを振とうまたは揺動すると、コラーゲンゲルの片側への細胞の不均一な分布になることがあります。 37℃インキュベーター内でプレートを配置します。 </li><li>細胞は、毎日のE培地を交換し、コンフルエントに成長する必要があります。細胞がコンフルエントにあるとき培地中に変更後の黄色の一日にメディア変更します。これは、通常2-4日間かかります。培地は一日4で変更されていない場合は、次のステップに進みます。 </li></ol> 4。ラフトの文化を作る<ol><li>滅菌した金属製のいかだのグリッドを配置するために滅菌ピンセットを使用して、側面が曲がっ、10cmディッシュに。コラーゲンゲルから培地を吸引除去する。井戸の両側からゲルを緩め、上下運動を用いた滅菌スパチュラを用いてゲルの周囲に移動します。コラーゲンゲルを削除するには、わずかに板を傾けて、ヘラの下に配置することによって持ち上げます。グリッドとゲルの間に気泡を発生させることなく、金属グリッド上にコラーゲンゲルを置きます。つのゲ​​ルは、各グリッドに配置することができます。 </li><li>気液界面を作るために、いかだのグリッドがメディアに触れているので、皿の底にゆっくりとEGFなしでE培地を追加しますが、コラーゲンゲルではありません。メディアは、グリッド内の穴を通って来るべきではありません。これは均一な分化を防ぐことができますので、グリッドの下に気泡が発生しないように徐々に培地を追加します。 37℃でラフトをインキュベートし、気液界面を維持し、一日おきに培地を変更します。 14日後にラフトを収穫。 </li></ol> 5。代表的な結果と、t彼プロトコルがプライマリケラチノサイトで正しく実行されている場合、この手順は、皮膚の異なる層が明白である高分化型上皮になります。これは、図1Bに示すように、ヘマトキシリン​​およびエオシン（H＆E）用のいかだ文化の切片を染色して組織学的検査によって達成される。 H＆E染色では、ラフトと階層化と分化する細胞の能力にウイルスや抗ウイルス化合物の影響の形態を調べるためにも有用である。たとえば、HPV-不死化ヒト一次ケラチノサイト（HFK-31）から作られたラフトは特に厚く増殖率の増加と分化細胞をアクティブに維持するためにHPVタンパク質の能力を反映して、正常なヒト包皮角化細胞（HFK）から作られたラフトと比較されます細胞周期（図1B）10インチ上皮全体に核の保持この結果、正常なケラチノサイトの破壊、その結果、分化に細胞周期を終了するのに対し、核膜。より密接に差別化を調べるために、免疫組織化学は、サイトケラチンと同様に、インボルクリン、およびフィラグリンなど、特定の分化マーカーを調べるために行うことができます。別のサイトケラチンは、分化7の特定の段階で表現されています。図2に示すように、サイトケラチン10（K10）は、基底層で表現されていない、とだけ徐々に分化細胞で構成される基底上の層に含まれています。 HPV陽性のラフトに、K10の発現が再びケラチノサイト分化のHPVタンパク質の影響を反映して正常なケラチノサイトのそれに比べて遅れている。免疫組織化学は、ウイルス遺伝子の発現プロファイルを調べるために使用することができます。図3に示すように、式上皮の最上位層に限定されている後半のタンパク質であるHPVタンパク質E1 ^ E4の発現を調べる代表的な染色です。予想通り、全く染色が正常フーマーからいかだ培養で観察されないnは包皮ケラチノサイト。 <img alt="図1" src="/files/ftp_upload/3668/3668fig1.jpg" /> 図主なケラチノサイトまたは上皮細胞から器官いかだ培養を調製するための方法の1）。概要。安定したHPV-31（HFK-31）エピソーム、または正常なヒト包皮角化細胞（HFK）からを維持するヒト包皮角化細胞から生成されたカドヘリン培養のB）、ヘマトキシリン​​およびエオシン染色。個々の上皮層が識別されるだけでなく、コラーゲンプラグインされています。 <img alt="図2" src="/files/ftp_upload/3668/3668fig2.jpg" /> 図2：サイトケラチン10（K10）の発現は上皮の基底層直上の層に限定されています。免疫組織化学は、HFK-31細胞と同様に、K10に対する抗体を使用して、通常のHFKsから生成されたカドヘリンの文化の断面を行った。細胞のDNAはDAPIで対比されました。画像は、confを使用してキャプチャされましたOCAL蛍光顕微鏡。矢印は上皮の基底層を示しています。 <img alt="図3" src="/files/ftp_upload/3668/3668fig3.jpg" /> 図3。HPV E1 ^ E4タンパク質が遅いウイルスのライフサイクルの生産段階で生成されます。免疫組織化学は、HFK-31細胞と同様に、E1E4に対する抗体を用いて通常のHFKsから生成されたカドヘリンの文化の断面を行った。細胞のDNAはDAPIで対比されました。画像は、共焦点蛍光顕微鏡を用いて捕獲された。矢印は上皮の基底層を示しています。

<ol>
	<li>Andrei, G., Duraffour, S., VandenOord, J., Snoeck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+raft+cultures+for+investigations+of+virus+growth,+pathogenesis+and+efficacy+of+antiviral+agents.">Epithelial raft cultures for investigations of virus growth, pathogenesis and efficacy of antiviral agents.</a> Antiviral Research. 85, 431-449 (2010).</li><li>Asselineau, D., Prunieras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+'simplified'+skin:+control+of+fabrication.">Reconstruction of 'simplified' skin: control of fabrication.</a> The British Journal of Dermatology. 111, 219-222 (1984).</li><li>Bodily, J., Alam, S., Chen, H., Meyers, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organotypic epithelial raft cultures and the study of the natural history of human papillomavirus. , (2006).</li><li>Cheng, S., Schmidt-Grimminger, D. C., Murant, T., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation-dependent+up-regulation+of+the+human+papillomavirus+E7+gene+reactivates+cellular+DNA+replication+in+suprabasal+differentiated+keratinocytes.">Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes.</a> Genes &#38; Development. 9, 2335-2349 (1995).</li><li>Dollard, S. C., Wilson, J. L., Demeter, L. M., Bonnez, W. R., Reichman, C., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+papillomavirus+and+modulation+of+the+infectious+program+in+epithelial+raft+cultures.+OFF.">Production of human papillomavirus and modulation of the infectious program in epithelial raft cultures. OFF.</a> Genes &#38; Development. 6, 1131-1142 (1992).</li><li>Frattini, M. G., Lim, H. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+synthesis+of+oncogenic+human+papillomaviruses+requires+episomal+genomes+for+differentiation-dependent+late+expression.">In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 3062-3067 (1996).</li><li>Kopan, R., Traska, G., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoids+as+important+regulators+of+terminal+differentiation:+examining+keratin+expression+in+individual+epidermal+cells+at+various+stages+of+keratinization.">Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization.</a> The Journal of Cell Biology. 105, 427-440 (1997).</li><li>Longworth, M. S., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+human+papillomaviruses+in+differentiating+epithelia.">Pathogenesis of human papillomaviruses in differentiating epithelia.</a> Microbiology and Molecular Biology Review. 68, 362-372 (2004).</li><li>Meyers, C., Frattini, M. G., Hudson, J. B., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+human+papillomavirus+from+a+continuous+cell+line+upon+epithelial+differentiation.">Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation.</a> Science. 257, 971-973 (1992).</li><li>Wilson, R., Laimins, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Differentiation of HPV-Containing Cells Using Organotypic Raft Culture or Methylcellulose. , 119-119 (2006).</li><li>zur Hausen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Papillomavirus+infections--a+major+cause+of+human+cancers.">Papillomavirus infections--a major cause of human cancers.</a> Biochimica et Biophysica Acta. 1288, F55-F78 (1996).</li></ol>

我々は、開示することは何もありません。

ここでは一般的には上皮分化を研究するために使用できるメソッドを記述するだけでなく、容易にウイルスの病原性と同様に、潜在的な治療薬の有効性を研究するために適応させることができます。多くのウイルスは、ヘルペスウイルスと同様に、HPVの場合のように、またはウイルスのライフサイクルのある時点で、感染症のプライマリサイトのいずれかとして上皮細胞を標的とする。宿主?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>ラット尾コラーゲン1型（4-5mg/ml） </td><td> BDバイオサイエンス</td><td> 354236 </td></tr><tr><td> NaHC0 3なしのDMEM </td><td>インビトロジェン</td><td> 12100-061 </td></tr><tr><td> NaHC0 3 </td><td>シグマ</td><td> S5761 </td></tr><tr><td> HEPES </td><td>カルビオケム</td><td> 391338 </td></tr><tr><td>ステンレス鋼の金属グリッド</td><td>ウィリアムズと気概（株） </td><td> CR-03063-040-100-S </td></tr><tr><td> E媒体11 </td><td></td><td></td></tr><tr><td>上皮成長因子</td><td> BDバイオサイエンス</td><td> 354010 </td></tr></tbody></table>

generation of organotypic raft cultures from primary human keratinocytes

器官上皮いかだの文化の発展は、in vitroの系を忠実に繰り返すという上皮分化に効率的に研究を提供しています。このシステムには多くの用途があります。例えば、ケラチノサイトの三次元カドヘリンの文化を成長させる能力は、ヒトパピローマウイルス（HPV）1の研究において重要なマイルストーンであった。 HPVのライフサイクルは、しっかりと扁平上皮の2分化にリンクされています。器官上皮いかだ培養は、ウイルス産生3,4,5を含む全体のパピローマウイルスのライフサイクルを再現ここで示された。さらに、これらのラフト文化はHPVを用いた in vivo感染の際に観察されたものと同様の異形成病変を示す。したがって、このシステムは、上皮細胞がんと同様に、一般に上皮細胞の分化に及ぼす薬物の効果を研究するために使用することができます。もともとAsselineauとPrunierasによって開発された<sアップ&gt; 6とKopan らによって変更されます。7、器官上皮いかだ培養システムは、コラーゲンに細胞の増殖を伴う一般的な、比較的容易に培養モデル、へと成長した気液界面（図1A）を維持し接続します。 10-14日の間に、細胞が層別化と差別化、分化特異的なケラチンを生成する完全な厚さの上皮を形成する。収穫ラフトと同様に標準的な分子生物学的および生化学的手法により、組織学的に調べることができます。本稿では、初代ヒトケラチノサイトからいかだ文化の生成方法を説明します。同じ手法が確立され上皮細胞株で使用でき、かつ容易に正常または病気の生検8から上皮組織での使用に適合させることができます。その複製のライフサイクルの一環として、多くのウイルスのターゲットのいずれかの皮膚または粘膜上皮。過去数年間、カドヘリンカルトを使用しての可能性ウイルス-宿主細胞の相互作用を研究する方法としてURESは、いくつかのヘルペスウイルスと同様に、アデノウイルス、パルボウイルス、およびポックスウイルス9に示されている。カドヘリンの文化は、このようにウイルスの病原性を調べるために適応し、永続的な細胞株での耕作ではありませんこれらのウイルスのための新規な抗ウイルス剤をテストする唯一の手段であることができます。

Watch this Scientific Journal Video about Generation of Organotypic Raft Cultures from Primary Human Keratinocytes at JoVE.com

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

不定冠詞<em&gt; in vitroで</em模倣する&gt;メソッド<emin vivoでの&gt;</em&gt;上皮分化が記載されている。多くのウイルスは、ウイルスのライフサイクルの一環として、上皮細胞を標的とし、この方法は、ウイルスを調べる手段を提供します。ホストの相互作用より密接に発生することに似ている<emin vivoでの&gt;</em&gt;。この技術は、主ケラチノサイト、確立された細胞株と同様に、正常なまたは病気の生検組織で使用することができます。

初代ヒトケラチノサイトからカドヘリン文化の生成

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

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Immunology and Infection

19.1K ビュー.  University of North Carolina-Chapel Hill. 不定冠詞 in vitroでメソッド上皮分化が記載されている。多くのウイルスは、ウイルスのライフサイクルの一環として、上皮細胞を標的とし、この方法は、ウイルスを調べる手段を提供します。ホストの相互作用より密接に発生することに似ている。この技術は、主ケラチノサイト、確立された...

ビデオ: Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

成人肝細胞の混合初代培養からマクロファージを分離するためにシンプルかつ効率的な方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

免疫・感染学

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

初代ヒトナイーブおよびメモリーT細胞から誘導した調節性T細胞の生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

新生仔ラットの脳組織の混合グリア細胞培養物から主要なミクログリアの分離

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

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 ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

末梢血からのヒトアロ抗原特異的T細胞の生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

不死化ソートB細胞からの組換えヒトIgGモノクローナル抗体の作製

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

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 ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

全血からのヒト単球由来樹状細胞の生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

マウスからの初代肝細胞培養における炎症反応の誘導

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

次世代エレクトロポレーションによって初代ヒトTh17細胞の小さなRNAのトランスフェクション

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...