この作品は、メイヨークリニック（CLH）から初期のキャリア開発賞で、NINDS（CLH）から助成金NS64571でサポートされており、ドナルドとフランシスHerdrich（CLH）からの寛大な贈り物がされました。我々は援助のためのメイヨークリニックのフローサイトメトリーコアを感謝したいと思います。

1。頭蓋内ウイルスの注入： 次のテクニックは変更と私たちの研究室と同僚によって広く利用されています。簡単に言うと、タイラーのマウス脳脊髄炎ウイルス（TMEV）または偽感染のダニエルの菌株の頭蓋内注射は、（1、10）脳浸潤白血球（BILs）を引き出すために若いマウス（年齢、好ましくは5-6週間）に実行されます。結果は、ダニエルの歪み、Beanのひずみ、およびGDVII株間で異なることに注意してください。 BILsを収穫する目的で、マウスはTMEVの2 × 10 5 PFUを受け取ると24時間感染している。 <ol><li>自動1ミリリットルハミルトンシリンジに、注射針（27ゲージ、¼ケンドール、で）添付して、ウイルスの10μlを提供するように設定。 </li><li>気泡に細心の注意とシリンジに10μLDMEMでTMEVを描く。適切な場合は、偽感染マウスは、ウイルスを含まないDMEM 10μlを受け取る。 </li><li>注射の直前に、下に綿の基板とワイヤグリッドを含むベルジャーにイソフルランの約1 mLを注ぐ。 </li><li>ベルジャーのワイヤグリッドの上に置いてマウスはdireclty彼らが軽く、麻酔つま先-ピンチに区別しないと吸入麻酔薬、約10〜15秒で固定化になるまで。物質が刺激性及び腐食することができるようにしたマウスは、イソフルランと直接接触しないでください。 </li><li>麻酔下にある間、素早くシェービングとインクを使用して頭蓋骨上前頭皮質領域で適切な座標を配置することにより、注射のためのマウスを準備する。注射のための一般的な場所は、ブレグマの前方に1 mmの右側の矢状縫合（図1）の横1mmである。定位注射は、いくつかの実験的な状況に適したかもしれないが、我々は、シェービングやインクなしでフリーハンドの注入が実験の大部分に適していることを発見した。両方の軸に沿って針先で毛皮を別れと注入に適しています。 </li><li>注入を行うには、オリエント約3 mmの深さに骨を介して頭蓋骨と記者に針の垂直。 </li><li>ハミルトンシリンジのボタンを押して撤退する前に脳を入力するようにウイルスのために2秒を待っていることにより脳内へのエクスプレスウイルス。 </li><li>慎重に針を除去し、清潔で乾燥したケージでマウスを置きます。マウスはすぐに麻酔から覚醒し、数分以内に正常な機能を再開する。動物の安楽死として、そのような注射部位から多量の出血のように、異常な症状を示すマウスに適切な注意を与えることが適切な場合があります。 6、健康と動物実験の国立研究所の遵守及び委員会のガイドラインを使用しての処理に適切な動物に従う必要があります。 </li></ol> 2。脳浸潤白血球細胞の準備を： <ol><li> 10ミリリットルRPMI 1640と30ミリリットルの容量を保持できるの丸底オークリッジ遠心管を準備し、9ミリリットルパーコールし、1mlの10 × PBSとチューブは、脳の削除処理中にベンチにRTに座っているはず。 </li><li>室温（RT）にをFicoll - Paqueプラスをもたらす。 </li><li>修正されたイソフルラン過剰摂取5で動物を安楽死させるとすぐに氷上で5ミリリットルRPMIを含む15 mlのコニカルチューブにステンレス鋼の組織のヘラと場所を使って脳を取り除く。ある状況下では、PBS -灌流はBILsの準備から、循環末梢血細胞を除去することが適切な場合があります。マウスは、完全かつ適切に麻酔前にPBS -灌流にしてください。 </li><li> 7ミリリットルガラスパイレックスブランドTenbroeck組織グラインダーに脳とRPMI液を移し、ゆっくりと10〜15ストロークでホモジナイズする。 </li><li> 2回アップダウンホモジナイザーとピペットにピペットで追加の5ミリリットルRPMIを。転送脳ホモジェネート（約10ml）を事前に準備された丸底チューブへと静かに転倒させて2〜3回がミックスする。 </li><li>ベックマンアレグラX - 22R臨床遠心分離機でF0360固定アングルローターで室温で30分7800グラムAveで脳ホモジネートを回転させる。 </li><li>遠心分離後、勾配の上に浮いているミエリンの残骸を削除します。赤血球細胞ペレット（図2）の上に浮いて白血球層を収集する。 </li><li> 50 mlコニカルに40μmのセルストレーナーを通して白血球層を負担。 RMPIで50mlの容積にソリューションを立ち上げます。 </li><li>室温で5分間1500rpmで（600グラムAVE）の臨床遠心機で希釈細胞懸濁液を含むチューブを回転させる。 </li><li>上清を吸引除去し、細胞ペレットを収集する。 1ミリリットルのFACS緩衝液（1％ウシ血清アルブミン、0.02％ナトリウムカルシウムのアジ化物とマグネシウムを含まないPBS）でペレットを再懸濁し、5 mlの丸底のFACSチューブに細胞懸濁液を移す。 </li><li> 1ミリリットルをFicoll - Paqueのプラスと慎重にアンダーサスペンション。 </li><li>ブレーキなしで室温で25分間臨床遠心機で2500rpmで（1400与えた）でチューブを回転させる。 </li><li> 1 mlのピ​​ペットと新しい5ミリリットルFACSチューブへの転送とのインタフェース（図2）に白、ふわふわのレイヤーを削除します。ウォッシュC4 mlのFACS緩衝液でエルズ。 </li><li>細胞をペレット化への臨床遠心分離機で室温で5分間1500rpmで（600グラムAVE）でチューブを回転させる。 </li><li>上清を吸引除去し、細胞ペレットを収集する。適切な緩衝液で白血球ペレットを再懸濁し、新しいFACSチューブには氷上に置きます。 </li><li>トリパンブルー排除により、細胞の生存率を評価し、フローサイトメトリーで分析し、細胞を数える。一般的には、研究者は約5 × 10 5細胞/動物を取得することを期待することができます。 </li></ol> 3。フローサイトメトリーによる免疫表現型検査<ol><li>白血球を分離し、計数した後、臨床遠心分離機で1500rpmで（600が与えた ）で3分間、細胞をスピン。 </li><li> ° Cで4℃30分間と10時05分01秒の割合でウシ胎児血清とインキュベートする。2.4G2ハイブリドーマ（anti-CD16/32のFcブロック）からの上清を、FACS緩衝液：以下を含むブロッキングバッファーでペレットを再懸濁し。 </li><li>準備し、4℃で30分間、1:200とインキュベートの濃度でブロックされた細胞·ダークのCに細胞外抗原に対する標識抗体を加える。 96穴V底プレートに200μlで細胞を染色し、インキュベートする。適切なコントロールが必要に応じて未染色細胞と蛍光色素補正のサンプルを、含まれています。 </li><li> 96ウェルプレート用ローターに適切なを使用して臨床遠心分離機で1500rpmで3分間室温で染色された細胞を（600 与えた ）スピン。 </li><li>上清を吸引除去し、穏やかに3回上下にピペッティングして200μlのFACS緩衝液で細胞を洗浄。 </li><li>二度、上記の洗浄技術を繰り返します。 </li><li>洗浄後、2％パラホルムアルデヒドおよびFACSチューブへの転送中に細胞を懸濁します。固定は、バイオセーフティーレベル2の試薬が感染した細胞のフローサイトメトリー解析のために必要です。 </li><li>修正されたフローサイトメトリー法8次のBD FACSキャリバーで固定された細胞を実行し、オフラインFlowJo、WinMDIまたは他の利用可能なフローサイトメトリー解析ソフトウェアを使用してファイルを分析する。 </li><li>サンプルあたり50-100,000イベントの分析は、一般的に十分な免疫表現型検査のために必要です。 </li></ol>この実験で使用されている直接標識抗体： CD45は、クローン30 F11で検出した。 Ly6C / GはクローンGR1、RB6 - 8C5で検出した。 CD11bは、クローンM1/70で検出された。 F4/80はクローンBM8で検出された。 4。代表的な結果： 私たちは24時間の感染後（図3）でマウスのBILsのための細胞の表現型解析の結果を示す。白血球は、炎症性単球を検出するために、PE標識抗マウスLy6C / G、APC標識抗マウスCD11bおよび細胞を検出するためにF4/80抗マウスAPC標識、免疫細胞を検出するためにPerCP結合抗マウスCD45で染色した単球系の。解析は、BD FACSキャリバーの楽器を行った。 <img src="/files/ftp_upload/2747/2747fig1.jpg" alt="図1"/> 図1。注射部位の解剖学的局在。注射部位は、ブレグマの前方に1 mmの右側の矢状縫合の外側を1mmに位置しています。 <img src="/files/ftp_upload/2747/2747fig2.jpg" alt="図2"/> 図2。白血球とBILsの勾配分離のイラスト。白血球は、最初は直接ミエリンの破片の層の下にとPercoll勾配でRBCペレット上に収集されます。界面で白い、ふわふわの層（BILs）はフィコール勾配から収集されます。 <img src="/files/ftp_upload/2747/2747fig3.jpg" alt="図3"/> 図3。 24時間後に感染。マウスの白血球における脳infilatrating白血球の免疫表現型は、組織が ​​頭蓋内感染24時間後に動物から調製したホモジネートの差密度遠心分離によって脳から単離された。細胞は、マウスCD45、Ly6C / G、CD11b、およびF4/80に対する蛍光標識抗体で染色した。最初のゲートは、前方散乱光（FSC）、セルサイズの指標（C）に対して、CD45、免疫細胞のマーカーをプロットすることにより行った。 CD45 HI（A、D）、CD45 LO（B、E）、およびCD45 NEG（F、G）の人口は、単球とマクロファージマーカーLy6C / G、F4/80（A、Bの相対的な発現レベルについて分析した、F）、およびCD11b（D、E、G）。 Ly6C / G + F4/80 + CD11b +炎症性単球は末梢からのこれらの細胞の浸潤と一致し、CD45 ハイテクの人口（A、D）でほとんど独占的に発見された。対照的に、Ly6C/G-F4/80 LO CD11b +マクロファージが常駐表現型と一致し、CD45 LO（B、E）とCD45 NEG（G）の集団でほぼ全面的に認められた。数々の実験では、我々はCD45 ハイテク細胞や非感染の脳または擬似感染のLy6C / G + F4/80 + CD11b +炎症性単球を観察したことがないテッドマウス（データは示さず）。

<ol>
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利害の衝突は宣言されません。

我々は日常的に脳浸潤細胞調製物の品質の両方を決定するためにフローサイトメトリーを使用し、免疫細胞2,9の異なる集団を区別する。急性の時間の時点で、私たちのBILsメソッドはCD45hi集団内の炎症性単球と同様にCD45lo集団におけるマクロファージの高い割合の高い割合を得られます。これは、脳内で再現性の免疫応答が確実に私たちの方法によって特徴付けることができることを?...

<table border="1"><tbody><tr><th>試薬</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td> TMEV </td><td>ハウラボ</td><td> N / </td><td></td></tr><tr><td>ダニエルの歪み</td><td>インビトロジェン</td><td> 21063-029 </td><td></td></tr><tr><td>イソフルレン</td><td> Novaplus </td><td> NDC 0409-3292-49 </td><td></td></tr><tr><td>丸底オークリッジ遠心管</td><td> Nalgene </td><td> 3118-0030 </td><td></td></tr><tr><td> RPMI 1640 </td><td>インビトロジェン</td><td> 11875-093 </td><td></td></tr><tr><td>パーコール</td><td> GEヘルスケア</td><td> 17-0891-02 </td><td></td></tr><tr><td> 10 × PBS </td><td>ロッシュ</td><td> 11666789001 </td><td></td></tr><tr><td>をFicoll - Paqueプラス</td><td> GEヘルスケア</td><td> 17-1440-02 </td><td></td></tr><tr><td>トリパンブルー0.4％（w / v）の</td><td> Mediatech </td><td> 25から900 - CI </td><td></td></tr><tr><td> 15 mlコニカルチューブ</td><td> BDファルコン</td><td> 352097 </td><td></td></tr><tr><td> 7 mLのガラスパイレックスブランドTenbroeck組織グラインダー</td><td>フィッシャー</td><td> 08から414 - 10B </td><td></td></tr><tr><td> 40μmのセルストレーナー</td><td> BDファルコン</td><td> 352340 </td><td></td></tr><tr><td> 50 mLコニカル</td><td> BDファルコン</td><td> 352070 </td><td></td></tr><tr><td>ウシ血清アルブミン</td><td>シグマ</td><td> A9647 </td><td></td></tr><tr><td>アジ化ナトリウム</td><td>シグマ</td><td> S8032 </td><td></td></tr><tr><td> CMF - PBS </td><td> Mediatech </td><td> 21から040 - CV </td><td></td></tr><tr><td> FACSチューブ</td><td> BDファルコン</td><td> 352054 </td><td></td></tr><tr><td>ウシ胎仔血清</td><td>シグマ</td><td> F4135 </td><td></td></tr><tr><td> 2.4G2ハイブリドーマ</td><td> ATCC </td><td> HB - 197 </td><td></td></tr><tr><td>コスターV -底板</td><td>コー​​ニング</td><td> 3894 </td><td></td></tr><tr><td>アレグラX - 22R遠心分離機または同等の</td><td>ベックマン</td><td> N / </td><td></td></tr><tr><td> 96ウェルプレートのバケットとローター</td><td>ベックマン</td><td> S2096 </td><td></td></tr><tr><td>アングルローター</td><td>ベックマン</td><td> F0360 </td><td></td></tr><tr><td>パラホルムアルデヒド</td><td>シグマ</td><td> P6148 </td><td></td></tr><tr><td> BD FACSキャリバー</td><td> BDバイオサイエンス</td><td> N / </td><td></td></tr><tr><td> FlowJo 7.5 </td><td>ツリースター株式会社</td><td> N / </td><td></td></tr><tr><td> CD45 </td><td> BDバイオサイエンス</td><td> 557235 </td><td>クローン：30 - F11 </td></tr><tr><td> GR1（Ly6C / G） </td><td> BDバイオサイエンス</td><td> 553128 </td><td>クローン：RB6 - 8C5 </td></tr><tr><td> CD11b </td><td> ebiosciences </td><td> 17-0112-83 </td><td>クローン：M1/70 </td></tr><tr><td> F4/80 </td><td> ebiosciences </td><td> 17-4801-82 </td><td>クローン：BM8 </td></tr></tbody></table>

isolation of brain-infiltrating leukocytes

我々は、マウスからの脳浸潤白血球（BILs）を調製するための方法を説明します。我々はどのように脳全体から浸潤細胞の白血球に富む集団を精製するために迅速な頭蓋内注入法とを経由してタイラーのマウス脳脊髄炎ウイルス（TMEV）をマウスに感染させる方法を示します。簡単に言えば、マウスは、密閉チャンバーのイソフルランで麻酔し、前頭皮質にハミルトンシリンジで注入フリーハンドです。マウスはその後Tenbroeck組織グラインダーを含むRPMIで抽出し、均質化されイソフルラン過剰摂取と全脳による感染後の様々な時点で殺されている。脳ホモジネートを、ミエリン及び他の細胞破片を除去するために、連続30％Percoll勾配を通じて遠心分離されています。細胞懸濁液を、40μmの時に緊張洗浄し、白血球を選択し、精製する不連続をFicoll - Paqueプラス勾配で遠心分離される。白血球を洗浄し、フローサイトメトリーで免疫表現するための適切な緩衝液に再懸濁する。フローサイトメトリーはC57BL / 6マウスにおける感染の初期段階での自然免疫細胞の集団を明らかにする。 24時間後に感染症で、免疫細胞の複数のサブセットにはGR1 +、CD11b +とF4/80 +細胞の濃縮の人口で、BILsに存在しています。したがって、この方法は、脳内の急性感染症に対する免疫応答を特徴づけるのに便利です。

Watch this Scientific Journal Video about Isolation of Brain-infiltrating Leukocytes at JoVE.com

Isolation of Brain-infiltrating Leukocytes

マウス脳から浸潤白血球を得るために迅速な方法が記載されている。この方法は連続Percoll勾配と白血球に富む層を選択し、精製する不連続フィコール勾配を利用しています。分離された白血球は、フローサイトメトリー測定によって特徴付けることができる。

脳浸潤白血球の分離

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

isolation-of-brain-infiltrating-leukocytes

Research

JoVE Journal

Immunology and Infection

19.0K ビュー.  Mayo Clinic College of Medicine. マウス脳から浸潤白血球を得るために迅速な方法が記載されている。この方法は連続Percoll勾配と白血球に富む層を選択し、精製する不連続フィコール勾配を利用しています。分離された白血球は、フローサイトメトリー測定によって特徴付けることができる。

ビデオ: Isolation of Brain-infiltrating Leukocytes

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

マウス腹腔細胞の分離

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

免疫・感染学

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

機能心臓の免疫細胞の単離

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

脳から隔離された白血球の単離と解析<em&gt;マラリアベルゲイ</em&gt; ANKA感染マウス

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

ヒト白血球と血小板の単離と評価のためのプロトコル：生体エネルギーと酸化バースト

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

フローから白血球の補充に間質細胞の影響を分析する

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

ヒト母体·胎児のインターフェイスから白血球の単離

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

母体·胎児界面におけるマウス組織からの白血球の分離

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

酵素消化および勾配分離を使用したマウス皮膚から浸潤白血球の単離

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating肺白血球の選択的ハーベスティング

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-肝白血球の選択的ハーベスティング

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...