Name: isolation and flow cytometric characterization of murine small intestinal lymphocytes
Uploaded: 2016-05-08T15:00:00.000+00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes at JoVE.com

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&amp;nbsp;</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&quot; 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&amp;nbsp;</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 &amp;mu;m Cell strainer</td><td>Falcon</td><td>08-771-19</td><td></td></tr><tr><td>40 &amp;mu;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

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 ...

免疫・感染学

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 ...

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 ...

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 ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

マウス胸腺、膵リンパ節および脾臓フローサイトメトリーを用いた排水における制御性 T 細胞サブセットの定量

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

マウスB細胞の発達におけるフロー細胞量測定

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

フローサイトメトリーによる分析のための子宮内生リンパ球細胞の分離

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

マウス小腸上皮オルガノイドと先天性リンパ球細胞との共培養

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

スペクトラルフローサイトメトリーにより固形組織から単離された細胞懸濁液の分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

生物学

The Isolation of Flowing Mesenteric Lymph in Mice to Quantify In Vivo Kinetics of Dietary Lipid Absorption and Chylomicron Secretion

Intestinal lipoproteins, especially triglyceride-rich chylomicrons, are a major driver of metabolism, inflammation, and cardiovascular diseases. However, isolating intestinal lipoproteins is very difficult in vivo because they are first secreted from the small intestine into the mesenteric lymphatics. Chylomicron-containing lymph then empties into the subclavian vein from the thoracic duct to deliver components of the meal to the heart, lungs, and, ultimately, whole-body circulation. Isolating na&#239;ve chylomicrons is impossible from blood since chylomicron triglyceride undergoes hydrolysis immediately upon interaction with lipoprotein lipase and other lipoprotein receptors in circulation. Therefore, the original 2-day lymph fistula procedure, described by Bollman et al. in rats, has historically been used to isolate fresh mesenteric lymph before its entry into the thoracic vein. That protocol has been improved upon and professionalized by the laboratory of Patrick Tso for the last 45 years, allowing for the analysis of these critical lipoproteins and secretions from the gut. The Tso lymph fistula procedure has now been updated and is presented here visually for the first time. This revised procedure is a single-day surgical technique for installing a duodenal feeding tube, cannulating the mesenteric lymph duct, and collecting lymph after a meal in conscious mice. The major benefits of these new techniques include the ability to reproducibly collect lymph from mice (which harnesses the power of genetic mouse models); the reduced anesthesia time for mice during the implantation of the duodenal infusion tube and the lymph cannula; the ability to continuously sample lymph throughout the feeding and post-prandial period; the ability to quantitatively measure hormones and cytokines before their dilution and enzymatic hydrolysis in blood; and the ability to collect large quantities of lymph for isolating intestinal lipoproteins. This technique is a powerful tool for directly and quantitatively measuring dietary nutrient absorption, intestinal lipoprotein synthesis, and chylomicron secretion.