JSOはNIHルースL.キルシュNRSA（F32 DK091040）によってサポートされて、AKは米国糖尿病協会（7-10-MI-05）からのポストドクトラルフェローシップによってサポートされており、AHHは、米国心臓協会設立研究者賞によってサポートされています（12EIA8270000）。フローサイトメトリー実験は、リソース共有VMCフローサイトメトリーで行った。 VMCフローサイトメトリーの共有リソースがバンダービルト消化器病研究センター（DK058404）によってサポートされています。

1。試薬および消耗品前にこの実験プロトコルを開始するには、次の試薬を準備します。 <ol><li> 70％エタノール</li><li> 1X PBS </li><li> 1X DPBSは（CaおよびMgを含まない）0.5％BSAを補っ</li><li> FACSバッファー：1X DPBS（CaおよびMgを含まない）、2mMのEDTA、および1％FCS </li><li> ACK緩衝液：150mMのNH 4 Clを、10mMのKHCO 3、および水中0.1 mMの硫酸ナトリウムEDTA </li></ol> 2。脂肪組織の収穫と準備<ol><li>各機関に固有のIACUC承認された手順に従って、マウスを安楽死させる。 </li><li>徹底的に70％エタノールで毛皮が濡れ。 </li><li>剣状突起のレベル（胸骨の下の部分）に切開を行い、心臓を露出する胸腔を開いて、すべての主要な血管を切断しないように注意しながら。 </li></ol> 注：この時点で、それはできるだけ絞りをそのままにしておくことも有用である可能と血液を可能にし、胸腔から流出する潅流液に胸郭の右側のノッチをカットする。 <ol start="4"><li>血を許可し、循環系をエスケープする潅流液に右心房をクリップ。 </li></ol> 注意：AT肥満マウス、過剰心を扱うときは、心臓へのアクセスを許可するために除去する必要があるかもしれません。 <ol start="5"><li>鉗子で心をつかみ、静かに頂点を通して左心室に針を挿入します。ゆっくり15ミリリットル滅菌PBSでマウスを灌流。 </li></ol> 注意：肺は記入して拡大し始めたら灌流率を減らします。 <ol start="6"><li>腹膜腔を開き、任意の性腺組織を避けるように注意して使用してperigonadal脂肪パッドを取り外します。 </li><li>の脂肪パッドを置きますと、0.5％BSAを補充し2ミリリットル1X DPBSをマグネシウム（Mg又はCaなし）を含む氷の上にボートを比較検討し、細かい部分にATをミンチ。 </li></ol>注：ATあたりの量を制限し、1.2 gのボートを量る。 AT量が1.2 gを超える場合、2つの計量船との間に均等に分割する。 <ol start="8"><li>氷の上にサンプルAT維持し、0.5％BSA、10mMの塩化カルシウム、及び4 mg / mlのコラゲナーゼ、II型で補充1X DPBSから成るサンプルAT当たり3ミリリットルコラゲナーゼ消化液を調製。 </li></ol> 3。コラゲナーゼ消化<ol><li>ホモジネートを注ぐと、1mlのDPBS（0.5％BSA）と3ミリリットルコラゲナーゼII消化液でボートを量るすすぐことによって、少なくとも50 mlコニカルチューブに移します。 </li><li> 20分間で37回転シェーカー（200 rpm）で°Cでホモジネートでインキュベートする。 </li><li>コニカルチューブや氷​​の上の場所に10ミリリットルのDPBSを（0.5％BSA）を追加します。 </li><li> 10ミリリットル血清ピペットを用いて何度もホモジネート摩砕し、新しい50 mlコニカルチューブに100μmのフィルターを介して細胞懸濁液を渡す。 </li><li> 4＆Dで10分間500×gで細胞懸濁液を遠心例えば、C. </li><li>混入赤血球を溶解する3ミリリットルACKバッファー内の上清をデカントし、再懸SVF細胞ペレット。 </li><li> 4℃で10分間500×gで、12ミリリットルのFACS緩衝液および遠心細胞懸濁​​液を追加℃で</li><li>上清をデカントし、FACSバッファーで再懸SVF細胞ペレット。 </li></ol> 注：細胞ペレットを50 mlコニカルチューブの底をカバーする場合、0.5〜1ミリリットルFACS緩衝液を使用するインチ再懸濁するためにどのくらいのFACS緩衝のためのガイドとして細胞ペレットのサイズを使用し、それ以外の場合、0.25から0.5に再懸濁ミリリットル。 <ol start="9"><li>場所氷上でサンプルと混合40μlのFACS緩衝液、50μlのトリパンブルー溶液（0.2％）、および10μlの細胞懸濁液で細胞計数のために、各サンプルの10倍希釈のアリコートを準備します。 </li><li>トリパンブルー排除に基づく生細胞をカウントし、5〜10×10 6細胞/ mlの最終濃度に細胞懸濁液を希釈。 </li></ol> 4。細胞表面抗原の染色<oL&gt; <li> 0.5-1μg/106細胞の最終濃度に抗マウスCD16/CD32抗体（FCブロック）を追加し、10分間、氷上でインキュベートする。 </li><li> 12×75ミリメートルポリスチレン丸底チューブへの転送サンプル（≥10 6細胞）。 ATMやT細胞を分析する場合、別々のチューブを用意し、必要な補償と修正蛍光マイナス1（FMO）コントロールに対応するために、チューブの適切な数を準備するために余分なセルを結合します。 </li></ol> 注：例として、F4/80とCD11bをに基づいてATMの割合を定量するとき、次の補償およびFMOコントロールが準備する必要があります。 - 染色（細胞） - DAPIまたはヨウ化プロピジウム（PI）は、単一の染色（細胞;ステップ5.1まで生存染料を追加しないでください） - F4/80 APC単染色（細胞や補償ビーズ） - CD11bをFITC（シミ単一細胞または補償ビーズ） - FMO 1（セル）：ラットIgG2aのκアイソタイプ対照APC + CD11bをFITC +生存色素（ステップ5.1で追加された） - FMO 2（セル）：F4/80 APC +ラットIgG2bでκアイソタイプ対照FITC +生存色素（ステップ5.1で追加された） <ol start="3"><li> （ 試薬および材料の表を参照）は、適切な濃度で蛍光団標識1次抗体および/ ​​またはアイソタイプコントロールを追加します。 </li><li> 30分間4℃で光とインキュベートからサンプルを保護します。 </li><li> 4℃で5分間500×gで2 mlのFACS緩衝液および遠心細胞懸濁​​液を追加℃で</li><li> 2ミリリットルFACS緩衝液で上清をデカントし、再懸SVF細胞ペレット。 </li><li> ≥400μlのFACS緩衝液で5 4℃分、再懸濁しSVF細胞ペレット用に細胞懸濁液を500×gで遠心します。 </li><li> 35μmのセルストレーナーチューブトップを装備し12×75ミリメートルポリスチレン丸底チューブにサンプルを転送します。</li><li> 4で光、店舗サンプルから守る°CをFACS分析まで。 </li></ol> 注：最適な結果を得るために細胞をimmeadiately分析する必要があるが、このプロトコルとFACS解析に成功し、4℃で1-2時間、FACSバッファーに格納された標識された細胞上で実施されている細胞は蓄積時間を増加させるために修正する必要がある場合は、標識した細胞をFACS分析の前に24時間、4℃で2％パラホルムアルデヒド°Cで固定することができる。抗体会社は、標識した細胞を1週間に戻る保存することができることを示唆しているが、これは、この手順のコンテキスト内でテストされていません。 5。 FACS分析<ol><li> FACS分析に先立ち、生/死細胞の区別を可能にするために、サンプルと適切なコントロールに生存染料を追加します。 </li></ol> 注：多数の生存色素は市販されているが、DAPIやPIは励起/発光教授に応じて推奨されている使用されるフ​​ルオロフォア標識抗体の諸島。 DAPIおよびヨウ化プロピジウムは、0.2 mg / mlの最終濃度を各サンプルに添加する。 <ol start="2"><li>関心のある細胞集団（s）がスケールであるように側方散乱（SSC）及び前方散乱（FSC）を調整するために、実験試料と同じ組織から単離された染色陰性対照試料、好ましくはセルを使用。 </li></ol> 注：SVF細胞ATの分析のために、SSCはリニアスケールでFSC対ログスケールで表示されることをお勧めします。しばしば非常に大きく、粒状であるATMのを分析する際にSSCのログ·スケールを使用することは特に重要です。 <ol start="3"><li>分析される細胞の種類（複数可）に基づいて初期散乱光ゲートを描き、染色細胞が遠く（約10 2を中心とする）は、単一のパラメータヒストグラムの左端になるように光電子増倍管（PMT）のゲインを調整する適切なチャネルのために。&lt;/李&gt; </ol> 注：これは、リンパ球やマクロファージ（または他の骨髄細胞）により、自家蛍光の違いに別々に分析することをお勧めします。 <ol start="4"><li>マルチカラー補正を実行するために、単一の染色されたコントロールや抗体捕捉補償ビーズを使用してください。 </li></ol> 注：補償ビーズの使用が推奨されますが、各抗体の互換性が確保されなければならない。例えば、補償ビーズが単離された細胞は、適切な単一シミ制御を得るために必要とされる場合には、ウサギ抗体と交差反応しないかもしれない。 <ol start="5"><li>修正されたFMOコントロールに基づく実験的ゲートを設定します。 </li><li>関心のある集団の有病率に基づいてイベントの適切​​な数を収集し、実験データを記録するためにフローサイトメトリーを設定する。 </li><li>オフライン分析用にエクスポートFCSデータファイル。フローcytometrための利用できる多数のプログラムがありますyのデータ解析。我々はCytobank、investigatoresを格納し、分析データを、インターネット回線18を持つ任意のコンピューターからの数字を生成することができ、Webベースのプラットフォームをお勧めします。さらに、Cytobankがアクセス可能なデータを公開するか、協力者へのアクセスを制限する機能を提供しています。 </li></ol>

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我々は、開示することは何もありません。

肥満の代謝結果における免疫系の役割への関心の高まりは、ATの免疫細胞を特徴付けるためにフローサイトメトリーの普及につながっている。正確なプロトコルは、独自の経験や利用可能な装置に基づいて研究室の間で異なりますが、重要なステップは、コラゲナーゼ消化、差遠心分離し、細胞表面抗原のラベルが含まれています。本記事の目的は、ATおよび/またはフローサイトメトリーを?...

歴史的には、脂肪組織（AT）は、エネルギーバランスに応じて伸縮する脂質貯蔵、不活性器官として見てきた。我々は今、ATが積極的に直接摂食行動と全身のグルコース恒常性に影響を与えるホルモンの数を分泌する動的な内分泌器官を表していることを理解しています。また、過去十年間の間質血管画分（SVF）と同様に、ATホメオスタシスへの貢献ATに存在する免疫細胞の多数の人口の増加感謝があった。 差動遠心し脂肪細胞とコラゲナーゼダイジェストを使用してSVF AT分離する能力は、最初1964年1 Rodbellによって記述されていた。コラゲナーゼIIは、ほとんどの場合、脂肪細胞のインスリン受容体1のメンテナンスのため、脂肪細胞とSVF分離のために使用される。 ATの早い段階で、酵素分画は主に脂肪を研究するために採用されました代謝をcyteと脂肪前駆細胞を分離する。最近になって、フローサイトメーターの普及状況と市販のフルオロフォア標識抗体の増え続けると組み合わせこの技術は、免疫細胞ATの特徴付けを容易にしている。 AT炎症における免疫細胞の存在は以前に2に記載されていたが、ワイズバーグらによる精液の論文。とXu ら 。 2003年に出版さは、炎症性サイトカインを分泌し、AT-特定および全身インスリン抵抗3,4と相関肥満におけるマクロファージ（ATMの）、ATの蓄積を文書化することが最初でした。これらの観察結果は、調査の新たな分野の基礎は最近、 &quot;immunometabolism、&quot; 5造語を務め、樹状細胞6、肥満細胞7、T細胞8を含む様々な免疫細胞集団を、関与研究によってフォローアップされています-10、Bセル11、NKT細胞12、好酸球13、および肥満関連したインスリン抵抗性の発達中の好中球14,15。 この記事の目的は、SVF ATの細胞を単離するATMなどの特性を、フローサイトメトリーを介してT細胞ATするために使用されるコラゲナーゼ消化技術の詳細な説明を提供することである。このプロトコルは、ATマウスに最適化されていますが、視聴者は16で、人間のために、この手法の最適化に関する広範な詳細を提供する優れた記事を読んでから利益を得ることができる。この記事の対象読者は、限られた経験を持つ研究者でマウスを操作すると、フローサイトメトリーを実行を含む。時間とリソースを持つ細胞の収量と生存能力のバランスをとるためのいくつかの実用的な考慮事項は、免疫細胞集団AT特徴付けるためだけでなく、最適なフローサイトメトリーコントロールとして提示される。我々のプロトコルに加えて、読者は言及せている適切なコントロールと補償17を含むように、フローサイトメトリーの技術的な側面のいくつかの優れた議論のためのBasuさんらによる最近のJoveの記事への ED。

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog #</td>
 <td></td>
 </tr>
 <tr>
 <td>DPBS (no Ca or Mg) 10 x 500 ml</td>
 <td>Life Technologies</td>
 <td>14190-250</td>
 <td></td>
 </tr>
 <tr>
 <td>DAPI</td>
 <td>Life Technologies</td>
 <td>D3571</td>
 <td></td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Sigma</td>
 <td>A2153-100G</td>
 <td></td>
 </tr>
 <tr>
 <td>collagenase</td>
 <td>Sigma</td>
 <td>C6885-5G</td>
 <td></td>
 </tr>
 <tr>
 <td>propidium iodide solution</td>
 <td>Sigma</td>
 <td>P4864-10 ml</td>
 <td></td>
 </tr>
 <tr>
 <td>stable stack 20 microliter</td>
 <td>Rainin</td>
 <td>SS-L10</td>
 <td></td>
 </tr>
 <tr>
 <td>20 microliter filter tips</td>
 <td>Rainin</td>
 <td>SRL10F</td>
 <td></td>
 </tr>
 <tr>
 <td>stable stack 250 microliter tips</td>
 <td>Rainin</td>
 <td>SS-L250</td>
 <td></td>
 </tr>
 <tr>
 <td>1000 microliter tips</td>
 <td>Rainin</td>
 <td>GPS-L1000</td>
 <td></td>
 </tr>
 <tr>
 <td>1000 microliter filter tips</td>
 <td>Rainin</td>
 <td>GP-L1000F</td>
 <td></td>
 </tr>
 <tr>
 <td>250 microliter Filter Tips</td>
 <td>Rainin</td>
 <td>SR-L200F</td>
 <td></td>
 </tr>
 <tr>
 <td>FC Block</td>
 <td>BD Biosciences</td>
 <td>553142</td>
 <td></td>
 </tr>
 <tr>
 <td>fisher 100mn strainers</td>
 <td>Fisherbrand</td>
 <td>22-363-549</td>
 <td></td>
 </tr>
 <tr>
 <td>medium weigh dish</td>
 <td>Fisherbrand</td>
 <td>02-202B</td>
 <td></td>
 </tr>
 <tr>
 <td>aluminum foil</td>
 <td>Fisherbrand</td>
 <td>1213100</td>
 <td></td>
 </tr>
 <tr>
 <td>mincing scissors</td>
 <td>Fisherbrand</td>
 <td>089531B</td>
 <td></td>
 </tr>
 <tr>
 <td>Vortex</td>
 <td>Fisherbrand</td>
 <td>2215365</td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml conical tubes</td>
 <td>BD Falcon</td>
 <td>14-959-49A</td>
 <td></td>
 </tr>
 <tr>
 <td>filter top FACS tubes</td>
 <td>BD Falcon</td>
 <td>352235</td>
 <td></td>
 </tr>
 <tr>
 <td>10 ml pipette case 200</td>
 <td>BD Falcon</td>
 <td>1367520</td>
 <td></td>
 </tr>
 <tr>
 <td>round bottom tubes</td>
 <td>BD Falcon</td>
 <td>352058</td>
 <td></td>
 </tr>
 <tr>
 <td>5 ml syringe</td>
 <td>BD Falcon</td>
 <td>309646</td>
 <td></td>
 </tr>
 <tr>
 <td>V Bottom Plates</td>
 <td>Costar</td>
 <td>07-200-107</td>
 <td></td>
 </tr>
 <tr>
 <td>transfer bulb pipette</td>
 <td>Thermo Scientific</td>
 <td>13-711-22</td>
 <td></td>
 </tr>
 <tr>
 <td>Shaker</td>
 <td>Thermo Scientific</td>
 <td>11 676 071</td>
 <td></td>
 </tr>
 <tr>
 <td>Adhesive mat</td>
 <td>Thermo Scientific</td>
 <td>1368750</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Culture Centrifuge</td>
 <td>Sorvall</td>
 <td>75253839</td>
 <td></td>
 </tr>
 <tr>
 <td>Adapters</td>
 <td>Sorvall</td>
 <td>75003723</td>
 <td></td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD16/CD32</td>
 <td>BD Biosciences</td>
 <td>553142</td>
 <td>Concentration: 0.5 - 1 &mu;g/ 106 cells</td>
 </tr>
 <tr>
 <td>Rat anti-mouse F4/80</td>
 <td>eBioscience</td>
 <td>17-4801</td>
 <td>Fluorophore conjugate: APC 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 17-4321</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD11b</td>
 <td>eBioscience</td>
 <td>11-0112</td>
 <td>Fluorophore conjugate: FITC 
 Concentration: 0.5 &mu;g/ 106 cells 
 Isotype control catalog number: 11/1/4031</td>
 </tr>
 <tr>
 <td>Armenian Hamster anti-mouse CD11c</td>
 <td>eBioscience</td>
 <td>12-0114</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.8 &mu;g/ 106 cells 
 Isotype control catalog number: Dec-88</td>
 </tr>
 <tr>
 <td>Goat anti-mouse MGL1/2</td>
 <td>R&amp;D Systems</td>
 <td>FAB4297P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC108P</td>
 </tr>
 <tr>
 <td>Goat anti-mouse CD206</td>
 <td>R&amp;D Systems</td>
 <td>FAB2535P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC108P</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CCR2</td>
 <td>R&amp;D Systems</td>
 <td>FAB5538P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC013P</td>
 </tr>
 <tr>
 <td>Armenian Hamster anti-mouse TCR&beta;</td>
 <td>BD Biosciences</td>
 <td>553174</td>
 <td>Fluorophore conjugate: APC 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 553956</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD8a</td>
 <td>BD Biosciences</td>
 <td>552877</td>
 <td>Fluorophore conjugate: PE-Cy7 
 Concentration: 0.8 &mu;g/ 106 cells 
 Isotype control catalog number: 552784</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD4</td>
 <td>BD Biosciences</td>
 <td>557956</td>
 <td>Fluorophore conjugate: Alexa Fluor 700 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 557963</td>
 </tr>
 </tbody>
</table>
Table 1. List of Materials and Reagents.

isolation of adipose tissue immune cells

肥満の齧歯類およびヒトの脂肪組織の増加マクロファージ浸潤（AT）の発見は、局所および全身インスリン抵抗性免疫細胞の寄与の関心の増大につながっている。リーンと肥満のさまざまな免疫細胞集団の単離と定量AT今immunometabolismの研究室で一般的に利用技術です。まだ細心の注意を得られたデータは、信頼性と解釈可能であるように間質血管細胞の分離やフローサイトメトリー分析の両方に注意しなければならない。このビデオでは、ダイジェストをミンチし、免疫細胞に富む間質血管の一部を分離する方法を示します。その後、我々は、抗体ラベルマクロファージとTリンパ球と方法、フローサイトメトリー実験でそれらに適切にゲートにする方法を示しています。低脂肪で育てリーン及び高脂肪給餌肥満マウスからの代表的フローサイトメトリープロットが設けられている。この分析の重要な要素は、何の抗体を使用することであるマクロファージで自然自家蛍光であるチャンネルだけでなく、適切な補償コントロールの使用で蛍光を発するではない。

差動遠心分離し、ATのコラゲナーゼ消化は、低脂肪（脂肪から10％キロカロリー）または高脂肪（脂肪から60％キロカロリー）食（LFDとHFDを与えた雄のC57BL/6Jマウスの精巣上体脂肪パッドからSVFを分離するために使用されました、それぞれ）16週間。 SVFの細胞は、その後、FACS解析により、生のATM（ 図1）およびT細胞（ 図2）AT割合を定量化するためにフルオロフォア標?...

Watch this Scientific Journal Video about Isolation of Adipose Tissue Immune Cells at JoVE.com

Isolation of Adipose Tissue Immune Cells

脂肪組織（AT）が激しい免疫細胞の活性化および相互作用の部位である。免疫系のほぼ全ての細胞は、ATに存在し、それらの比は肥満によって変更される。免疫細胞集団ATの適切な分離、定量化、および特性はimmunometabolic病気で自分の役割を理解するために重要である。

脂肪組織の免疫細胞の単離

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Research

JoVE Journal

Immunology and Infection

36.0K ビュー.  Vanderbilt University School of Medicine. 脂肪組織（AT）が激しい免疫細胞の活性化および相互作用の部位である。免疫系のほぼ全ての細胞は、ATに存在し、それらの比は肥満によって変更される。免疫細胞集団ATの適切な分離、定量化、および特性はimmunometabolic病気で自分の役割を理解するために重要である。

ビデオ: Isolation of Adipose Tissue Immune Cells

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

フローサイトメトリーで固定し、生細胞におけるカルパイン活性の測定

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

免疫・感染学

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

細胞星状に7つのステップ

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

マウス大動脈内の免疫細胞のフローサイトメトリー分析

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

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 ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

2光子顕微鏡を用いてマウスの胸腺の生体内イメージング

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

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 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, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

単離、特徴付けおよび歯肉免疫細胞ネットワークの機能検討

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Reliable and High Efficiency Extraction of Kidney Immune Cells

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

腎、免疫細胞の信頼性と高効率抽出

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

分離、拡張、および CD31 cd34 陽性 + + ひと大網と皮下脂肪組織から血管内皮細胞の脂肪分化誘導

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...