NKS is supported by NIH award K08 AI108690.

すべての研究は、実験動物学のためのアメリカ協会（AALAS）によって設定された獣医の基準を満たしているハーバード・メディカルスクールの施設内動物管理使用委員会（IACUC）に従って厳格な審査とガイドライン下で行いました。 共同住宅（オプション）を介して細菌の1水平伝播<ol><li>食品との両方がオートクレーブ処理された水を使用して、無菌の使い捨てケージを組み立てながら（ノトバイオートマウスを用いた場合は特に）外因性の汚染を最小限に抑えるために、無菌テクニックを練習。 </li><li>異なるmicrobiotasを保有し、同じケージでそれらを収容する6週齢のC57BL / 6マウスを個別にマークするために、耳パンチまたはタグを使用してください。マウスは食糞であり、ケージの床から糞を食べることを考えると、微生物が自然にマウスの間で水平方向に送信されます。 </li><li> ≥1週の共同住宅マウスは、分析前に微生物の転送および免疫学的変化のための時間を可能にします。 </li></オール&gt; 小腸上皮内のレイヤおよび固有層からの単一細胞懸濁液の調製<ol><li> 溶液の調製 <ol><li> （小腸あたり）抽出媒体の準備：30ミリリットルRPMI + 93μlの5％をジチオスレイトール（DTT）（w / v）の+ 60μlの0.5 M EDTA + 500μlのウシ胎児血清（FBS）。使用直前にDTTを追加します。 </li><li> （小腸あたり）消化メディアを準備：25ミリリットルRPMI + 12.5 mgのディスパーゼ+ 37.5 mgのコラゲナーゼII + 300μlのFBS。使用直前にディスパーゼおよびコラゲナーゼを追加します。 </li><li> 37°C、37°C​​にprewarmソリューションで行うすべてのインキュベーションのために。 </li></ol></li><li>頸椎脱臼に続いてCO 2窒息によりマウスを安楽死させます。 </li><li>ダウンマウスの背側に配置し、70％エタノールで腹部をスプレー。順次、あちこち腹側正中線に沿って皮膚、その後、腹膜筋膜を切断することにより開腹術を実行するためにハサミを使用して、このようにして腹腔を露出させ、剣状突起に恥骨結合をメートル。 </li><li>幽門括約筋を横断することにより、胃から小腸を分離するためにハサミを使用してください。ゆっくり腸間膜脂肪を離れてからかって、腹膜から小腸を削除します。 <ol><li>完全に小腸を削除するには、回盲接合部で第二切断してください。寒冷に分離小腸を配置し（ すなわち 、4℃）、10％FBSを含むRPMIに細胞生存率を最大にするために。 </li></ol></li><li>緩やかに湾曲鉗子を使用して、脂肪の大部分を削除します。脂肪を除去しようとしたときに、腸組織自体を引き裂くないように注意してください。 </li><li>注射器に取り付けられた18 G送り針を使用して冷PBSの20ミリリットル - 15で腸内容、優しくフラッシュ腸を削除します。 </li><li> PPを切除するためにハサミを使用し、5％FBSを含む冷RPMIに入れます。 PPSは、小腸のantimesenteric側に位置し、多分葉白として表示されます質量。 12のPP - 特定の菌株に応じて、マウスは、通常は8を持っています。 </li><li> 4インチのセグメント - 3に小腸をカット。 </li><li>離れて組織から脂肪をいじめるために鈍いメスを用いて、RPMIで湿らせたペーパータオル上の各小腸セグメントを圧延することにより、残留脂肪を取り除きます。 </li><li>湾曲した鉗子で腸セグメントをカニューレを挿入し、組織の遠位端をつかんで裏返し組織を回します。そして、そっと反転されている組織で、その結果、湾曲した鉗子（近位端で始まる）から組織セグメントを削除するには、ストレートピンセットを使用しています。 </li><li> 30抽出媒体のmlおよび撹拌棒を含むカップに組織セグメントを配置します。カップの蓋を固定し、そして37℃で15分間500rpmで攪拌​​。攪拌を激しくなく、乱流ではないでなければなりません。 </li><li>インキュベーション後、曇って表示されます上皮含有上清から組織片を分離するために鋼製のストレーナーを使用します。この上清は、私が含まれています2.23 - 2.18手順でサンプルを濾過するに進むことにより、所望の場合、さらに、分析することができるntraepithelialリンパ。 </li><li>手動での残留抽出媒体を洗い流すためにRPMIで組織片を攪拌します。 </li><li>乾いたペーパータオルで組織を置き、抽出媒体によって遊離されなかった残余の粘液の除去を容易にするために、端部の上に数回を終了フリップ。 </li><li>消化培地600μlの1.5 mlチューブに組織片を置きます。 </li><li>作品はもはやハサミに固執せず、溶液が均一表示されるまで、組織をミンチするはさみを使用してください。このステップでは、組織の完全な酵素消化を確保することが重要です。 </li><li>消化メディアの25ミリリットルを含むカップにみじん切り小腸を追加します。 37℃で30分間、500rpmで攪拌​​します。途中で消化（ すなわち 、15分で）を介して、アップピペットおよびダウン血清学的ピペットで組織の任意の大きな塊を破壊するために役立ちます。 </li><li>フィルタ消化された組織（またはepith50ミリリットルチューブに100μmのセルストレーナーを介して処理IELs場合はステップ2.12からelial層）。 10％FBSを含むRPMI 20mlでストレーナーを洗浄します。 </li><li> 4℃で10分間、500×gでろ過した溶液を遠心。 </li><li>慎重に10％FBSを含むRPMIの1ミリリットルで上清と再懸濁ペレットをデカント。 </li><li>フィルターを50mlチューブに40μmのセルストレーナーを介して細胞を再懸濁しました。 10％FBSを含むRPMI 20mlでストレーナーを洗浄します。 </li><li>遠心分離機は、4℃で10分間500×gで解決策を濾過しました。 </li><li>慎重に、2％FBSを含むRPMIの1ミリリットルで上清、およびペレットを再懸濁しデカント。 </li></ol>パイエル板からの単一細胞懸濁液の調製<ol><li>転送は25消化メディアのmlおよび撹拌棒をカップにのPPを切除しました。セキュアな蓋、および37℃で10分間、500 rpmでスピン。 </li><li>フィルターを50mlチューブに40μmのセルストレーナーを通してのPPを消化しました。任意の塊のREMA場合で、1mlシリンジからプランジャの平らな端を使用して、ストレーナを介してそれらを押してください。 </li><li> 10％FBSを含むRPMI 10mlでストレーナーを洗浄します。 </li><li>遠心分離機は、4℃で10分間500×gで解決策を濾過しました。 </li><li>慎重に、2％FBSを含むRPMIの1ミリリットルで上清、およびペレットを再懸濁しデカント。 </li></ol>フローサイトメトリー用4.表面染色<ol><li>細胞のアリコート適切な量（ 例えば 、LPLsの200μl）を96ウェル丸底プレートに。 </li><li> 4℃で5分間、500×gで遠心分離します。プレートを反転させて上清を除去。 </li><li> RPMI、2％FBSを含む90μlの再懸濁細胞と1：抗CD16 / 32（FCブロック）の100倍希釈。 4℃で10分間インキュベートします。 </li><li> 10μlのPBSを追加します。 4℃で5分間、500×gで遠心分離します。プレートを反転させて上清を除去。 </li><li> 250μlのPBSで細胞を再懸濁し。 4℃で5分間、500×gで遠心分離します。により上清を除去プレートを反転します。 </li><li>生存性染料と（オプション）ステイン細胞、必要に応じて、製造業者のプロトコルに従うことによって。 </li><li>細胞を固定するために、100μlの1％ホルマリンで細胞を再懸濁します。 4℃の暗所で1時間インキュベートします。 </li><li> 200μlのPBSを追加します。 4℃で5分間、500×gで遠心分離します。プレートを反転させて上清を除去。 </li><li> 250μlのPBSで細胞を再懸濁し。 4℃で5分間、500×gで遠心分離します。プレートを反転させて上清を除去。 </li><li> 200μlのPBSで細胞を再懸濁し。フローサイトメーターを用いて細胞を分析します。 </li></ol>

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No conflicts of interest declared.

我々はLPLs、IELs、およびPP内のリンパ球を含む小腸リンパ球の単離およびフローサイトメトリーの特性評価のためのプロトコルを提示します。微生物叢の変化が小腸管免疫系にどのように影響するかを評価することに興味のある方のために、私たちの詳細異なるmicrobiotasを保有するマウスとの間の生物の水平伝達に関与する簡単な手順。このプロトコルは、小腸に焦点を当てているが、手順が?...

小腸の主なタスクは、しばしば、栄養素1の消化吸収であると考えられます。この代謝機能が明らかに必須であるが、小腸内腔2内に見られる環境抗原の継続的な弾幕からホストを保護するのに等しく重要な役割を担っています。腸管外の世界を分離する（ 例えば 、管腔抗原）のみの単一細胞層の厚さである上皮層を持つホストの内部環境から。このように、小腸免疫系は、病原体の侵入に対して強固な応答を実装しながら、食事や共生微生物からの外来抗原があれば、最小限の、免疫応答と粘膜を入力することができ、反応性についてそのしきい値をバランスの手ごわい課題を有しており、その他の「有害な」抗原。これらの抗原に対する過剰または不適切な免疫応答は、病理学的疾患（ 例えば 、inflammaにつながることができます保守党腸疾患、I型糖尿病、多発性硬化症）と3-6を避けなければなりません。 全体的に、消化管は、すべて抗体分泌細胞7 70％以上含有し、体内で最大の免疫器官を表すと考えられています。粘膜固有層（LP）、上皮内層、およびパイエル板（PPS） - -小腸免疫系は、主に3つの区画で構成され、それぞれが、リンパ球2の独特のグループが含まれていること。 LPリンパ球（LPLs）は、主〜20％のB細胞とTCRαβ+ T細胞です。上皮内リンパ球（IELs）はTCRαβ+ T細胞よりもTCRγδ+ T細胞と非常に少数のB細胞が含まれています。そして小腸壁に埋め込まれた二次リンパ器官であるPPSは、〜80％のB細胞を含みます。これらの解剖学的領域のそれぞれはわずかに異なる機能と存在論的基盤を有しているが、彼らはああで機能します病原性の侮辱からホストを保護するためにファッションをarmonized。 さらに、微生物は、特定の微生物および特定の細胞系譜8,9の個体発生の間の同族関係の増加の認識で、腸管免疫系の開発のための重要な決定因子であることが成長の感謝があります。また、腸管免疫系の教育は、解剖学的に離れた部位（ 例えば 、関節炎、多発性硬化症、肺炎）で免疫応答に影響を与えることを考えると、それは以前に10を認識よりも、腸の免疫システムの開発は、より疾患プロセスに関連していることが明らかになってきました-12。同様にそのようなものとして、定量的に腸管免疫系を評価する上での関心は今ホスト共生相互作用を含むように宿主 - 病原体相互作用を超えて拡張しており、多くの全身性疾患の病因。 で現在の方法の変動性を考えます腸管リンパ球の分離、必要な時間のバランスを取ることはますます重要である一方で、収率、生存性、および一貫性のために最適化された方法。パーコール勾配が可変収量と生存率を13につながる、時間と労働集約的でヒューマンエラーへの潜在的になりやすいです伴うプロトコル。ここで、我々はすべての3小腸免疫コンパートメントからのリンパ球の単離および特徴付けのために最適化されたプロトコルを提供します。さらに、粘膜免疫系における微生物誘発性の変化関心の高まりを考えると、我々はこれらの変化を定量的に腸管免疫系にどのように影響するかを評価するためのマウスとの間の微生物の水平方向の伝送を可能にするために使用することができるステップが含まれます。

<table>
	<tbody>
		<tr>
			<td>Sterile Gloves</td>
			<td>Kimberly-Clark</td>
			<td>555092</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile mouse cage</td>
			<td>Innovive</td>
			<td>MS2-AD</td>
			<td>contains lid, cage bottom, and alpha-dri bedding</td>
		</tr>
		<tr>
			<td>metal feeder</td>
			<td>Innovive</td>
			<td>M-FEED</td>
			<td></td>
		</tr>
		<tr>
			<td>water bottle</td>
			<td>Innovive</td>
			<td>M-WB-300</td>
			<td></td>
		</tr>
		<tr>
			<td>card holder</td>
			<td>Innovive</td>
			<td>CRD-HLD-H</td>
			<td></td>
		</tr>
		<tr>
			<td>autoclavable rodent chow (NIH-31M)</td>
			<td>Zeigler</td>
			<td>4131207530</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>D5545-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA (pH 8.0)</td>
			<td>Ambion</td>
			<td>AM9262</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum (FBS)</td>
			<td>GemBio</td>
			<td>100-510</td>
			<td></td>
		</tr>
		<tr>
			<td>dispase II</td>
			<td>Invitrogen</td>
			<td>17105-041</td>
			<td>the concentration in the protocol is based on an activity level of 1.878 U/mg</td>
		</tr>
		<tr>
			<td>collagenase, type II&#160;</td>
			<td>Invitrogen</td>
			<td>17101-015</td>
			<td>the concentration in the protocol is based on an activity level of 245 U/mg</td>
		</tr>
		<tr>
			<td>dissecting scissors</td>
			<td>Roboz</td>
			<td>RS-5882</td>
			<td></td>
		</tr>
		<tr>
			<td>feeding needle (18 G, 2&#34; length)</td>
			<td>Roboz</td>
			<td>FN-7905</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml syringe</td>
			<td>BD</td>
			<td>305482</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpel (15 blade)</td>
			<td>Miltex</td>
			<td>4-415</td>
			<td></td>
		</tr>
		<tr>
			<td>curved forceps</td>
			<td>Roboz</td>
			<td>RS-5211</td>
			<td></td>
		</tr>
		<tr>
			<td>straight forceps</td>
			<td>Roboz</td>
			<td>RS-5132</td>
			<td></td>
		</tr>
		<tr>
			<td>multi-purpose cups, 120 ml</td>
			<td>VWR</td>
			<td>89009-662</td>
			<td></td>
		</tr>
		<tr>
			<td>stir bar</td>
			<td>VWR</td>
			<td>58949-062</td>
			<td></td>
		</tr>
		<tr>
			<td>multi-position stir plate, 9-position</td>
			<td>VWR</td>
			<td>12621-048</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel conical strainer, 3 inch&#160;</td>
			<td>RSVP</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml tube</td>
			<td>Eppendorf</td>
			<td>0030 125.150</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;m cell strainer</td>
			<td>Falcon</td>
			<td>08-771-19</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m cell strainer</td>
			<td>Falcon</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate, round-bottom</td>
			<td>Corning</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD16/32 (Fc block)</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td></td>
		</tr>
		<tr>
			<td>(optional) fixable viability dye eFluor 780</td>
			<td>eBiosciences</td>
			<td>65-0865-18</td>
			<td></td>
		</tr>
		<tr>
			<td>10% formalin, neutral buffered</td>
			<td>Thermo Scientific</td>
			<td>5725</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and flow cytometric characterization of murine small intestinal lymphocytes

腸 - 体内のあらゆる器官の免疫細胞の最大数が含まれている - 常に外来抗原、微生物および栄養の両方にさらされています。これらの管腔の抗原は、免疫応答を形作る助け、腸内の免疫細胞の教育が全身性疾患の数のために重要であることを増加理解を考えると、腸管免疫系を特徴付ける関心が高まっています。しかし、多くの公開されたプロトコルは、困難かつ時間がかかります。我々は、迅速な再現性があり、かつ面倒なパーコール勾配を必要としない小腸粘膜固有層、上皮内層、およびパイエル板からのリンパ球の単離のためにここに簡略化されたプロトコルを提示します。プロトコルは、小腸に焦点を当てているが、それはまた、結腸の分析に適しています。さらに、我々は特定の科学的QUESに応じて追加の最適化を必要とするかもしれないいくつかの側面を強調表示しましたる。このアプローチは、その後、フローサイトメトリー分析または特徴付けの代替手段を使用することができる生存可能なリンパ球の大量の単離をもたらします。

小腸リンパ球の単一細胞懸濁液のフローサイトメトリー分析は、脾臓細胞（ 図1Aおよび図1B）のような前方類似した側方散乱特性を有する細胞の個別の集団をもたらすべきです。リンパ球は組織が ​​低い前方散乱を有し、他の上皮細胞および死細胞（ 図1C）から分離するのがより困難であるリンパ球集団を生じる、分離の初期段階の間4℃?...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes at JoVE.com

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

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

マウスの小腸リンパ球の単離およびフローサイトメトリーキャラクタリゼーション

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

isolation-flow-cytometric-characterization-murine-small-intestinal

Research

JoVE Journal

Immunology and Infection

27.3K ビュー.  Boston Children’s Hospital. There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization. 

ビデオ: Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

機能と分子生物学的研究のためのプライマリマウスII型肺胞上皮細胞のフローサイトメトリ分離

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

免疫・感染学

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

マウス生殖管粘膜からのリンパ球の単離

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

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 ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

虚血性マウス脳からの免疫細胞の単離およびフローサイトメトリー分析

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

神経膠腫浸潤末梢血単核細胞の単離およびフローサイトメトリー分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Activation of Murine Lymphocytes

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

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

単離とマウスリンパ球の活性化

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

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

PM10における粒子結合のBet V 1アレルゲンのフローサイトメトリー分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

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 ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

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

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

マウス小腸管免疫系からリンパ球を分離すること

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

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.