この作品は、NIHのPBS（R 21 AI095484）によってサポートされています。私たちは、CD1dの四量体のために薬価四量体中核施設に感謝します。

1。楽器の調製、培養培地および試薬<ol><li>楽器：無菌条件下で試薬を準備し、層流フードの下で使用しています。 </li><li>ウシ胎児血清5％、炭酸水素塩2.1gを10mlのグルタミン酸、100倍、HEPESを10mg、1mgのピルビン酸ナトリウム及び10mlの100倍のペニシリン/ストレプトマイシンを追加し、RPMI組織培地を1リットル調製する。 注：推奨される組織培養培地、RPMIは、DMEMと交換可能である。 </li><li> 5％のコラゲナーゼ &quot;D&quot;溶液（組織培養培地9mlにコラゲナーゼ5mlの「D」）を調製する。 </li><li>三つの異なる濃度でのパーコール溶液を作る：100％、80％および40％であった。以下の濃度を、1サンプルについて計算される。室温でのソリューションにしてください。 <ol><li>パーコール液（原液から）希釈されていないパーコールの9ミリリットルを追加します。 PBSを10倍の1ミリリットルを加える。 </li><li>パーコール液：パーコール100パーセントの8ミリリットルを追加します。 PBS 1Xの2ミリリットルを追加します。 </li><li>パーコール液：パーコール80パーセントの4ミリリットルを追加します。 PBS 1Xの4ミリリットルを追加。 </li></ol></li><li> （FACS）のバッファを蛍光活性化細胞選別の1リットルを作る。 5ミリリットルの10％BSA（0.1％BSA）1mlの10％アジ化ナトリウム（0.1％）2mlのEDTA剤は、1×PBSで1000 mlに構成し、追加する。 </li></ol>腎臓消化の2。準備<ol><li>必要な制度の承認を取得した、犠牲と放血マウスを標準的な方法で、現在の規制や動物のケアのガイドラインに従うこと。 <ol><li>ペントバルビタール（0.07 mg / kg）をして、マウスを麻酔。 </li><li>放血に進みます。 </li><li>手術台上の仰臥位でマウスを置きます。 </li><li> 70％エタノールで腹部をスプレーしてください。 </li></ol></li><li>ペトリ皿にコラゲナーゼD溶液10mlを加える。 </li><li>各マウスから両方の腎臓を削除し、カプセル化解除。 <ol><li>胸骨からに腹部の皮膚および腹膜を通して正中切開を行うことによってabdomimal空洞を開く恥骨。 </li><li>側に横方向に腸に移動することにより、左の腎臓を公開します。 </li><li>慎重にピンセットで左のカプセルを分離する。 注：カプセルは腎臓の周囲の透明層として表示され、部分的に腎周囲脂肪組織でカバー。 </li><li>外科はさみを使用して椎弓血管を切断することにより、左の腎臓を削除します。 </li><li>右腎に対して同じ手順を繰り返します。 注：右腎より頭蓋正中（小花柄が短い）に近い。 </li></ol></li><li>ペトリ皿に腎臓を置きます。 </li><li>定期的な金属成形ブレードを用いて小片に腎組織（寸法1〜2 mm）をカットします（ 図1）37℃のインキュベーター内で30〜45分間コラゲナーゼ溶液にピースを置きます。 </li></ol>腎臓消化から腎臓単核細胞（KMNC）の3。準備<ol><li>腎臓の細胞懸濁液を得機械的に組織培養培地25ml中にストレーナ（70ミクロン）を用いて再懸濁し、混合組織を破壊することによる消化。 </li><li>細胞懸濁液をペレット化し、4℃で10分間、400×gで遠心する。 </li><li>非常にゆっくりと慎重に、40％パーコール溶液4mlでペレットを再懸濁し、穏やかに転送プラスチックピペットを用いて80％のパーコール溶液4mlに重ねる。これは明らかに、半透明の層によって分離された2つの相をもたらすであろう。一番上の脂質に対応する黄色の層となる。 </li><li>ブレーキオフモードで遠心分離して、室温で30分間、1,500×gで遠心分離する。 </li><li>吸引によって、黄色、厚いトップ層（約1〜2 ml）を外します。 </li><li>トランスファーピペットで2相の界面からのわずかな白っぽい半透明の層を収集します。非常に慎重に組織培養培地2mlを含有する15ミリリットルの円錐管を減らすこの懸濁液を転送し、その後の全量を13mlの培地を加える15ミリリットル。 </li><li> 4℃で10分間400×gで遠心分離</li><li>培地の1ミリリットルのサンプルを懸濁します。 </li><li>血球計数器でパンブルー排除を用いてKMNCの数をカウントし、腎臓あたりのml当たりKMNCの数字として結果を表現しています。 </li><li>上記のように一度洗う。 </li></ol>造血の4。分離KMNCから（CD45 +）細胞（ステップI） <ol><li>ランニング緩衝液90μlの中に10 7に細胞を再懸濁します。 </li><li> CD45マイクロビーズを10μlを加え、4℃で15分間インキュベート</li><li>反応を停止させ、ランニングバッファーの1ミリリットルを追加します。ランニング緩衝液500μlに4℃の再懸濁で10分間400×gで遠心します。 </li><li>磁石で配置することで、磁気カラムを準備し、ランニング緩衝液3mlで洗い流してください。 </li><li>磁気カラムを通して細胞懸濁液をパスし、カラムを通過する非標識細胞を収集します。 </li><li> runnの3ミリリットルで細胞を3回洗浄するバッファをING。その後、流出物を収集します。 </li><li>セパレータとピペットカラムにランニング緩衝液の5ミリリットルから列を削除し、すぐにラベルされた分を洗い流す。この画分は、CD45 +細胞が含まれています。細胞を数える。 </li><li>腎臓への異なるT細胞サブセットの絶対数を計算するには、フローサイトメトリーによって決定さ陽性細胞の割合（ 図2）によってKMNCの総数を乗算する。 </li></ol>ネガティブ選択によりCD45陽性の準備から、DN T細胞の5。アイソレーション（工程II） <ol><li>以下のビオチン化抗体の各々2.5μlの（0.5 mg / mlの、1.25μgの）追加：抗CD4、抗CD8、抗Fc受容体（CD16/32）を、抗MHCクラスII（IA b）は、抗すべての10 7造血細胞のためのCD1d PBS-57四量体。 </li><li>低温室で30分間細胞をインキュベートします。 </li><li> 1ミリリットル抗ビオチンマイクロビーズを追加します。 </li><li>低温室で30分間インキュベートします。 </li><li> Tチューブを配置彼は、磁石と枯渇に進みます。 </li><li>血球計数器を用いて陰性画分中のDN T細胞の総数を決定するために、生存細胞を数える。 </li><li>第一ゲートのCD45 +、第2ゲートTCRαβ+：このゲーティング戦略に従うことによって、フローサイトメトリーで、DN T細胞の純度を確認してください。 CD1dの+細胞を除外します。 DN T細胞（ 図3）を識別するためのプロットCD8 +対CD4 +。 </li><li>純度で決まる割合で合計数を掛けます。 注：90％以上純粋なDN T細胞の集団におけるこのプロトコルの結果を用いて。 </li></ol>

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著者らは、開示することは何もありません。

彼らはそのような自己免疫疾患、癌、移植性、および急性腎障害（AKI）を含む腎臓の原疾患、糸球体腎炎8、13の異なる病態に関与していることから、DN T細胞中の関心が高まっている。したがって、より良好なDN T細胞の病態生理学的機能を理解し、特徴付ける必要がある。しかしながら、現在CD4およびCD8 T細胞と比較して、これらの細胞の機能を理解する上で不足している。主な理由?...

A correction was made to: <a href="http://www.jove.com/video/51192/isolation-of-double-negative-t-cells-from-the-kidney">Isolation of Double Negative &#945;&#946; T Cells from the Kidney</a>.
There was an error with an author's name. The author's given name had a typo, this was corrected to:
Samatha Bandapelle
instead of:
Samantha Bandapelle

A correction was made to: Isolation of Double Negative &#945;&#946; T Cells from the Kidney.

Erratum: Isolation of Double Negative &#945;&#946; T Cells from the Kidney

周辺αβTCR+ CD3 + CD4 - CD8 -ダ ​​ブルネガティブ（DN）T細胞は、明確な表現型を有し、1〜4機能の様々な部分集合に分割される。 DN T細胞は、あまり理解されていますが、ますます異なる疾患モデル4-6の病態生理学的な免疫応答に関与している。 DN T細胞は、ますます様々な免疫および自己免疫応答の調節および同種移植寛容の調節に関与しているユニークな免疫細胞型である。4-6、9彼らは、末梢血および二次リンパ器官（脾臓及びリンパ節ではまれである）。しかし、彼らは正常な腎臓や腸上皮10月12日の主な居住者である。非常に少ないが、定常状態で、そのような腎臓transplに関連した急性腎障害（AKI）のような病理学的条件の下で、腎臓における機能7についてはほとんど知られていないANT。 なぜならalloresponsesを含む免疫応答を調節する上で様々な免疫細胞の複雑な役割のため、各プレイヤーの役割を定義するalloresponsesを理解し、新しい治療法を設計する上で非常に重要です。生理学的および異なる疾患状態の下で腎臓中に存在するDN T細胞の有意な数が指定され、DN T細胞は、移植レシピエントにおける免疫および自己免疫マウスおよびヒトにおける応答、およびalloresponsesの調節に重要な役割を果たしている可能性が高い。まだ散乱証拠かかわら蓄積すると、両方の病原性および免疫抑制機能のDN T細胞を関与しかし、なぜ、どのように特定の有害または抑制機能を発揮し、環境がそれらにどのように影響するかは十分に理解されていない。 により腎臓での少量に、分離する改良された方法が必要である。 Tから、DN T細胞を単離するための標準プロトコルが現在ありません彼は、非リンパ組織。我々のプロトコルは、腎臓からDN T細胞の単離のための新規な方法を記載している;しかしながら、この方法はまた、種々の非リンパ系組織のために使用することができる。

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			<td height="21" style="height:21px;width:439px;">Laminar flow hood&#160;</td>
			<td style="width:257px;">Baker Company, Inc</td>
			<td style="width:276px;"></td>
			<td style="width:200px;">Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Centrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Hemocytometer</td>
			<td>Electron Microscopy Sciences</td>
			<td style="width:276px;">63514-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Atmosphere-controlled incubator</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td style="width:200px;">&#160;(37&#176;C with 5% CO2)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Microscope&#160;</td>
			<td>Olympus</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Analytical flow cytometer</td>
			<td style="width:257px;">LSR II&#160;</td>
			<td></td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:439px;">Name of the reagent</td>
			<td style="width:257px;">Company&#160;</td>
			<td style="width:276px;">Catalog&#160; Number</td>
			<td style="width:200px;">Comments&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:439px;">RPMI 1640</td>
			<td style="width:257px;">Media Tech</td>
			<td style="width:276px;">10-040-CV</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Collagenase D</td>
			<td style="width:257px;">Roche</td>
			<td style="width:276px;">11088858001</td>
			<td style="width:200px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Percoll&#160;</td>
			<td style="width:257px;">GE Healthcare</td>
			<td style="width:276px;">&#160;17-0891-01</td>
			<td style="width:200px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Fetal bovine serum&#160;</td>
			<td>Corning Cellgro</td>
			<td>35-011-CV</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">0.1% sodium azide (NaN3)</td>
			<td style="width:257px;">Sigma-Aldrich&#160;</td>
			<td style="width:276px;">S2002</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Buffer phosphate buffered saline (PBS)</td>
			<td style="width:257px;">Corning Cellgro</td>
			<td>21-040-CV</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">PBS 10X</td>
			<td>Mediatech</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">EDTA buffer&#160;</td>
			<td style="width:257px;">Sigma-Aldrich&#160;</td>
			<td style="width:276px;">E1161</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">LS columns&#160;</td>
			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">CD45+ microbeads</td>
			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">6.78E-51</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Biotin microbeads&#160;</td>
			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">130-090-485</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD45 (clone: 30-F11)&#160; PerCP&#160;</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td style="width:276px;">G1397</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD45 (clone: 30-F11)&#160; APC-Cy7</td>
			<td style="width:257px;">Biolegend&#160;&#160;</td>
			<td style="width:276px;">103116</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-TCR Pacific Blue&#160; (ab-chain, clone: H57-597)</td>
			<td style="width:257px;">Invitrogen</td>
			<td style="width:276px;">HM3628</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD4 PE</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td>12-0043</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD8 FITC</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td>8011-0087</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD4 biotinylated (GK1.5)</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553728</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD8 biotinylated&#160; (clone 53-6.7)</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553029</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Fc receptor (CD16/32) biotinylated</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553143</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-MHC class II (I-A d) biotinylated</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553609</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD1d PBS-57 tetramer</td>
			<td style="width:257px;">NHI tetramer&#160;</td>
			<td style="width:276px;">lote # 15621-PBS57</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:439px;">AutoMACS Running Buffer-&#160; MACS Separation Buffer&#160;</td>
			<td style="width:257px;">MIlteny I Biotec</td>
			<td style="width:276px;">130-091-221</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">C57BL/6J mice</td>
			<td>Jackson Labs</td>
			<td style="width:276px;">000664</td>
			<td>Kidneys - Lymph Nodes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Petri dish&#160;</td>
			<td>BD Biosciences</td>
			<td style="width:276px;">356517</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">70mc filter&#160;</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Plunger&#160;</td>
			<td style="width:257px;"></td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GeneMate Tubes 50 ml</td>
			<td>Bioexpress</td>
			<td>C-3394-3</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">GeneMate Tubes 15 ml</td>
			<td>Bioexpress</td>
			<td style="width:276px;">C-3394-1</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Petri Dish&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:276px;">S33580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Plate 24 wells&#160;</td>
			<td style="width:257px;">BD Biosciences</td>
			<td style="width:276px;">354723</td>
			<td style="width:200px;"></td>
		</tr>
	</tbody>
</table>

isolation of double negative &#945;&#946; t cells from the kidney

非リンパ組織から、DN T細胞を単離するための標準的なプロトコルは、さまざまな免疫応答でのますます報告された関与にもかかわらず、現在ありません。 DN T細胞はallotransplants 1-6に免疫および自己免疫応答と寛容の調節に関与しているユニークな免疫細胞型である。 DN T細胞は、しかしながら、末梢血および二次リンパ器官（脾臓およびリンパ節）において稀であるが、正常な腎臓の主要な居住者である。非常に少ない起因周辺での不足への病態生理学的機能7についてはほとんど知られていない。我々は最近、定常状態でおよび虚血再灌流障害時に腎臓8でこの集団の包括的表現型および機能解析を説明した。 DN T細胞機能の分析が大幅に腎臓からのそれらの単離のためのプロトコルを開発することによって強化される。 ここでは、isolatioを可能にする新たなプロトコルを記述マウス腎臓由来の高純度AB CD4 + CD8 + T細胞およびDN T細胞のnである。手短に言えば、我々は、コラゲナーゼを用いて腎組織を消化し、密度勾配によって腎臓単核細胞（KMNC）を単離する。これはKMNCから3％〜70％の造血T細胞を濃縮するために二つのステップが続く。最初のステップは、CD45 +単離キットを使用して、造血細胞の正の選択で構成されています。第二段階において、DN T細胞は負にCD4、CD8、およびMHCクラスIIモノクローナル抗体およびCD1dのα-GalCer類似四量体を用いた非所望の細胞の除去によって単離される。この戦略は、90％以上純粋なDN T細胞の集団をもたらす。 FACS分析に続いて、上述した抗体による表面染色は、純度を確認するために使用される。

野生型C57BL / 6（B6）腎臓は、腎臓あたり約1.5から2.1×10 6の単核細胞が含まれています。約10％未満は、造血CD45 +細胞である。 5％のコラゲナーゼを用いて消化し ​​、続いて、図1に示すように、腎臓単核細胞（KMNC）の調製のために、腎臓を小片に切断する。 これはKMNC層（図2、左パネル）を収集する密度勾配遠心分離を行うこと?...

Watch this Scientific Journal Video about Isolation of Double Negative ÃŽÂ±ÃŽÂ² T Cells from the Kidney at JoVE.com

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

DNabT細胞は、末梢T細胞の中で稀であり;しかし、彼らは、特定の非リンパ組織に豊富に存在する。非リンパ組織からDN T細胞を単離することの難しさを認識病態生理学的有意性を増加させるにもかかわらず、それらの機能解析を妨げる。我々は、マウスの腎臓からの高度に精製されたDN T細胞の単離のための新規なプロトコルを記載している。

腎臓からの二重否定αβT細胞の分離

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

isolation-of-double-negative-t-cells-from-the-kidney

Research

JoVE Journal

Immunology and Infection

12.1K ビュー.  Johns Hopkins University School of Medicine.  DNabT細胞は、末梢T細胞の中で稀であり;しかし、彼らは、特定の非リンパ組織に豊富に存在する。非リンパ組織からDN T細胞を単離することの難しさを認識病態生理学的有意性を増加させるにもかかわらず、それらの機能解析を妨げる。我々は、マウスの腎臓からの高度に精製されたDN T細胞の単離のための新規なプロトコルを記載している。 

ビデオ: Isolation of Double Negative αβ T Cells from the Kidney

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 ...

免疫・感染学

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

マウスの腸からの樹状細胞およびマクロファージの単離とキャラクタリゼーション

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

臍帯血から前駆B細胞サブセットの分離

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

離とナイーブCD4 Tリンパ球のTh17分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

T細胞の導出<em&gt;体外</em&gt;マウス胚性幹細胞からの

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

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 ...

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&#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 ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T細胞は、樹状細胞からTransinfectionによって細菌をキャプチャ

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

ヒトパラティーノ扁桃腺からBおよびTリンパ球の効率的な単離プロトコール

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

ラット精巣からのセルトリ細胞と尿細管周囲細胞の単離

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...