Name: isolation of mouse coronary endothelial cells
Uploaded: 2016-07-03T13:00:00.000+00:00
Duration: 7 min 36 s
Description: Watch this Scientific Journal Video about Isolation of Mouse Coronary Endothelial Cells at JoVE.com

この作品は、国立衛生研究所（A.牧野へHL115578）からの助成金によってサポートされていました。

マウスでの研究は、アリゾナ大学の施設内動物管理使用委員会（IACUC）によって承認され、国立衛生研究所（NIH）のガイドラインに従って行きました。 注：セクション1、2および3は、セットアップとして、実験の前日に行われるべきです。 1.準備ソリューション<ol><li> CD31バッファー（50ml）で<ol><li> 50ミリリットルチューブに50ミリリットルの1×リン酸緩衝生理食塩水（PBS）を追加します。 0.05 gでウシ血清アルブミン（BSA）および0.037 gでエチレンジアミン四酢酸（EDTA）を秤量し、50mlチューブに追加し、粉末が溶解するまで約1時間回転するチューブを置きます。 、pHを7.4に調整し、新しい50ミリリットルチューブに0.2μmのフィルターでろ過し、使用するまで4℃で保存してください。 </li></ol></li><li> HEPESを有するハンクス平衡塩溶液（HBSS）（500mL）で<ol><li> HBSS溶液に10mMのHEPESを作り、よく混合するHEPESの1.1915グラムを追加します。 pH値を測定し、7.4に調整します。 4におけるフィルタと店舗˚C。 </li></ol></li><li> 1×クレブス溶液（1 L） <ol><li> 1リットルのフラスコに蒸留水を倍増する10×クレブス液[ 表1]の100ミリリットルを追加します。 2.1グラム重炭酸ナトリウムおよび2グラムD-グルコースを計量し、フラスコに追加します。混合し、1 Lに全量を</li><li>フードの内側30分およびフィルタソリューションの95％O 2/5％CO 2ガスとバブルソリューション。 4℃で保存し、実験中氷上に保ちます。 </li></ol></li></ol> 2.磁気ビーズを準備<ol><li>各1.5mlチューブ（サンプルあたり1管）に1ミリリットルCD31バッファを追加します。ヒツジ抗ラットIgGビーズを混合し、各サンプル[ 表1]に適切な量を追加します。勢いよく30回チューブを振ると揺れの1分間磁石板に配置します。プレート上にしながら、チューブを開き、バケットにソリューションをダンプします。 </li><li>各チューブに1ミリリットルCD31バッファを追加します。 2μlのラット抗マウスCD31抗追加します4ºCでチューブO / Nを各チューブにボディと回転します。 </li></ol> 3.オートクレーブ<ol><li>すべての手術ツールやピペットチップをオートクレーブ。 4直径の異なるヒントがあるように狭いために、最も広いから、ピペットチップの先端をカットします。ヒントのためのおおよその直径はそれぞれ3ミリメートル、2.5ミリメートル、2ミリメートル、および1.5ミリメートルです。ヒントをカットし、121ºCの温度で殺菌の30分に前日オートクレーブ。 注：セクション4から11は、実験の日に行われるべきです。 </li></ol> 4.準備ソリューション<ol><li> M199で2％の鉄を強化した胎仔血清（IFCS）を含む洗浄バッファーを準備します。イメージングのために、30ミリリットル/マウスを準備します。 </li><li>酵素液（10ミリリットル/サンプル）を準備します。 50ミリリットルチューブに0.6単位/ディスパーゼのミリリットルおよび1mg /コラゲナーゼタイプIIのミリリットルを計量。 M199を追加し、15分間、4ºCでチューブを回転させます。 insid新しい50ミリリットルチューブに0.2μmのフィルターでろ過電子フード、使用するまで4℃に保ちます。 </li><li>ヘパリンでHBSS / HEPESを準備します。 15ミリリットルチューブに10 mMのHEPESをHBSSの10ミリリットルを追加します。チューブにヘパリンの100μlを添加して、使用するまで4℃に保ちます。 </li></ol> 5.ビーズを洗浄<ol><li> 4ºCで回転子からビーズとチューブを取り出し、フードにもたらします。磁性板にチューブを置き、1分間振ります。ソリューションをダンプし、各チューブに1ミリリットルCD31バッファを追加します。 </li><li>磁性板の外管を取り、激しく30回振ります。磁性板に管をバック入れて、合計3回の洗浄のために繰り返します。終了後、4℃に戻し管を入れ、使用するまで回転し続けます。 </li></ol> 6.解剖<ol><li>血液凝固を防止するためにヘパリンを0.1 mlをマウスの腹腔内に注入します。 10分待ってから、ペントバルビタールの0.01ミリリットルの腹腔内注射を使用して、マウスを麻酔。マウスが感じているかどうかを確認します足をつまんで痛み。 </li><li>頭を上にしてポリスチレンボード上にマウスを置き、ピンと腕を保持します。喉まで胸部上部の領域で皮膚をカットする鉗子とタフなカットはさみを使用してください。 </li><li>ちょうど横隔膜上記の胸郭の中央にカットしてください。心臓や肺を露出する三角形を作るために喉に向かって横向きや上向きに骨をカットします。ちょうど横隔膜の上行大動脈をカットします。 </li><li>引っ張らずにピンセットで胸腺を持ち、一緒に肺と心臓を除去するために慎重に任意の結合組織を切断するはさみを使用しています。 1×クレブスを入れたビーカーに入れ組織は、鉗子で振ると1×クレブスで6センチメートル皿に移します。 </li><li>ハサミやピンセットを用いて肺葉を削除します。大動脈に20 G滅菌カテーテルを挿入します。 HBSS /ヘパリンと冠状動脈循環系に血液をフラッシュします。心室の小さなカットを行い、M199のサンプルチューブに心臓や転送を振ります。氷の上にチューブを保管してください。 </li></ol><p<html>クラス= &quot;jove_title&quot;&gt; 7。組織消化<ol><li>酵素液10mlを充填した6センチメートル皿に心を移し、はさみを使用して小片にそれをミンチ。 15分間、37ºCインキュベーターに皿を置きます。組織が容易に通過できるようにするために切断端と先端を通って下方に30回ピペッティングによって消化された材料を攪拌します。 </li><li>さらに15分間37ºCインキュベーターでそれを元に戻します。攪拌を3回繰り返して、最大の開口部からピペットチップを使用して、各時間が狭いと、それぞれの間に15分間インキュベーションします。 </li><li>最後の15分間のインキュベーション後、アウトとフードにサンプルを取ります。ピペット溶液を上下に30回、最も狭い開口部を有するピペットチップを使用して、新しい50ミリリットルチューブに40μmのセルストレーナーを通過します。 </li><li> 10mLのピ​​ペットを用いて10mLの洗浄緩衝液で皿を洗浄し、セルストレーナーに移します。 RTで10分間、400gで遠心分離し、スピンに50ミリリットルチューブを入れてください。 </li></ol>ビーズとitle &quot;&gt; 8。洗濯と共役<ol><li>ペレットを中断することなく、上清を取り除きます。二回5ミリリットル洗浄バッファーで細胞を洗浄。 </li><li>以前の1は、一度に1つのチューブにビーズや仕事1.5 mlチューブを用意しましょう​​。 、磁性板で1 1.5mlチューブを入れて3回振ると、それを開きます。 </li><li>最後の遠心分離後、ペレットを中断することなく、できるだけ多くの上清を除去します。積極的にフリックすることにより細胞塊の関連付けを解除します。 </li><li> 50mlチューブに1mLの洗浄緩衝液を追加し、1 mlピペットで細胞を混合します。 1.5ミリリットルチューブからソリューションをダンプし、ビーズに50ミリリットルチューブから細胞懸濁液の1ミリリットルを追加します。チューブを30回フリック、その後30分間、4ºCで回転します。 </li></ol> 9.細胞培養/イメージングのための準備<ol><li>細胞をプレーティングするためのチャンバーを準備します。表面もコーティングするためにチャンバーにゼラチン溶液（w / vの5％、300μl）を加え、37ºCで30分間インキュベートします。インキュベーション後、REMオベゼラチン及び乾燥した表面。 </li><li>回転の30分後にビーズ/細胞とチューブを取り出し、フード内部の磁気プレートに入れました。 2分、ダンプのために振ると1ミリリットルの洗浄緩衝液を追加します。 </li><li>プレートの外にチューブを取り、勢いよく30回、その後フリック30回振ります。磁性板でそれらを元に戻し、1分間振ります。 5回の洗浄の合計4回繰り返したが、1分がプレートに振盪しながら。 </li><li>最後の洗浄後、1チューブを取り出して、時に各チューブ1に取り組んでいます。その後、30回フリック30回振ります。磁性板の上に置き、1分間振ります。ダンプと1ミリリットル20％IFCS ECのメディアを追加（37ºCで予め温めておきました）。 </li><li>勢いよく30回、その後フリック30回振ります。 500μlのピペットを上下に10回ピペットを調整します。各チャンバーに500μlのを転送します。 </li><li> 37ºCインキュベーターに入れて、O / Nを残します。翌日、10％FBS EC培地で細胞を洗浄。 </li></ol>ウェスタンブロット（WB）サンプルについて10の準備<ol><li>ターク回転の1時間後にビーズ/細胞とチューブを電子。磁気プレートに入れ、2分間振ります。上清をダンプし、冷HBSS 1ミリリットルを追加します。勢いよく30回、その後フリック30回振ります。 3回の洗浄のために二度繰り返しますが、1分がプレートに振盪しながら。 </li><li>最後の洗浄後、磁性板にチューブを入れて、1分間振ります。ピペットを使用してバッファを削除し、以前に11,12を説明した方法を用いて、WBのための細胞を溶解します。収集された溶解物を、通常は約30μgのタンパク質のが含まれています。 </li></ol> 11.イメージング<ol><li>チャンバー250μlの最終容量で、メディアで細胞を3回洗浄します。細胞へのDil-acLDLの1.5μlを添加します。この点から、暗闇の中で細胞を維持します。 </li><li> 37℃で3.5時間インキュベートします。 、ヘキストの1.5μLを加え、よく混ぜて、PBSで37˚C.Wash細胞で30分間インキュベートし、室温で20分間、4％パラホルムアルデヒド（PFA）で固定します。 </li><li> PBSで2回洗浄し、30マイル、5％BSA-PBSでブロックRTでのn。 1％BSA-PBSで2回洗浄します。 2μg/ mlの濃度に1％BSA-PBSを用いてBS-レクチン-FITCを希釈し、各チャンバーに300μlを添加します。 </li><li>室温で30分間オービタルシェーカー上室を置きます。 PBSで3回洗浄します。 </li><li> FITC（レクチン染色、ECマーカー、励起/発光の488nm / 519 nm）を、DAPI（ヘキスト、358 nmでの核染色、励起/発光/ 30ミリ秒200ミリ秒の露光時間で蛍光顕微鏡下で細胞を可視化461 nm）であり、557 nmの/ 576 nm）におけるTRITC（acLDL染色、ECマーカー、励起/発光のための300ミリ秒。 20X対物レンズを使用して画像を取得します。 </li></ol>

<ol>
	<li>Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+functions.">Endothelial cell functions.</a> J Cell Physiol. 196, 430-443 (2003).</li><li>Sumpio, B. E., Riley, J. T., Dardik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+in+focus:+endothelial+cell.">Cells in focus: endothelial cell.</a> Int J Biochem Cell Biol. 34, 1508-1512 (2002).</li><li>Cines, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+in+physiology+and+in+the+pathophysiology+of+vascular+disorders.">Endothelial cells in physiology and in the pathophysiology of vascular disorders.</a> Blood. 91, 3527-3561 (1998).</li><li>Deanfield, J. E., Halcox, J. P., Rabelink, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+function+and+dysfunction:+testing+and+clinical+relevance.">Endothelial function and dysfunction: testing and clinical relevance.</a> Circulation. 115, 1285-1295 (2007).</li><li>Jin, Y., Liu, Y., Antonyak, M., Peng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+vascular+endothelial+cells+from+murine+heart+and+lung.">Isolation and characterization of vascular endothelial cells from murine heart and lung.</a> Methods Mol Biol. 843, 147-154 (2012).</li><li>Makino, A., Platoshyn, O., Suarez, J., Yuan, J. X., Dillmann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downregulation+of+connexin40+is+associated+with+coronary+endothelial+cell+dysfunction+in+streptozotocin-induced+diabetic+mice.">Downregulation of connexin40 is associated with coronary endothelial cell dysfunction in streptozotocin-induced diabetic mice.</a> Am J Physiol Cell Physiol. 295, C221-C230 (2008).</li><li>Marelli-Berg, F. M., Peek, E., Lidington, E. A., Stauss, H. J., Lechler, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+murine+tissue.">Isolation of endothelial cells from murine tissue.</a> J Immunol Methods. 244, 205-215 (2000).</li><li>van Beijnum, J. R., Rousch, M., Castermans, K., vander Linden, E., Griffioen, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+fresh+tissues.">Isolation of endothelial cells from fresh tissues.</a> Nat Protoc. 3, 1085-1091 (2008).</li><li>Nolan, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+signatures+of+tissue-specific+microvascular+endothelial+cell+heterogeneity+in+organ+maintenance+and+regeneration.">Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.</a> Dev Cell. 26, 204-219 (2013).</li><li>Sobczak, M., Dargatz, J., Chrzanowska-Wodnicka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+pulmonary+endothelial+cells+from+neonatal+mice.">Isolation and culture of pulmonary endothelial cells from neonatal mice.</a> J Vis Exp. , (2010).</li><li>Makino, A., Dai, A., Han, Y., Youssef, K. D., Wang, W., Donthamsetty, R., Scott, B. T., Wang, H., Dillmann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=O-GlcNAcase+overexpression+reverses+coronary+endothelial+cell+dysfunction+in+type+1+diabetic+mice.">O-GlcNAcase overexpression reverses coronary endothelial cell dysfunction in type 1 diabetic mice.</a> Am J Physiol Cell Physiol. 309, C593-C599 (2015).</li><li>Sasaki, K., Donthamsetty, R., Heldak, M., Cho, Y., Scott, B. T., Makino, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VDAC:+old+protein+with+new+roles+in+diabetes.">VDAC: old protein with new roles in diabetes.</a> Am J Physiol Cell Physiol. 303, C1055-C1060 (2012).</li></ol>

The authors have nothing to disclose.

研究において最も広く使用されている内皮細胞は、その利便性と培養の容易さのヒト臍帯静脈内皮細胞（HUVEC）です。しかし、多くの研究は、他の特定の臓器10からの内皮細胞の単離によって達成生理学的モデルの可用性を必要とします。内皮細胞は、心臓組織内の細胞の非常にマイナーな人口を構成しています。それらの単離および培養は非常に困難であることを証明することがで?...

様々な心血管疾患および代謝性疾患のマウスモデルは、患者に見られるものと類似している生理学的および分子的変化を負います。さらに、マウスの遺伝的変化は、私たちが疾患の発症と進行における特定の遺伝子の病原性の役割を調査することを可能にする強力なツールです。今日では、細胞型特異的遺伝子ノックインまたはチェックアウトマウスは簡単に多くの研究室で生成され、特定の細胞型におけるmRNAおよびタンパク質レベルの測定は、マウスは本当に遺伝的に変更されたかどうかを決定するための最初のステップであるされています。 内皮は、内皮細胞（EC）のラインの内側血管壁の薄い単層です。内皮細胞は、血管緊張、血管透過性、および新しい血管の形成1,2の調節に重要な役割を果たしています。内皮機能障害は、多くの病的状態および内皮機能の変化の顕著な特徴は、様々な車につながる可能性がありますdiovascular疾患3,4。生理学的および病態生理学的条件下での内皮細胞の機能を研究することが重要です。 電気部品5-8と分離法が使用される組織および種に応じて最適化する必要がある単離するためのいくつかの方法があります。異なる組織からのECは、それらの表面マーカーおよびタンパク質発現9に対して異質の高いレベルを表示するためです。内皮細胞の成功の分離は、しばしば挑戦することと訓練と実践のいくつかの学位を必要とすることができます。達成されると、このプロトコルで提案された単離方法は、安定的かつ効率的であることがわかります。 この方法の全体的な目標は、高品質と量のマウス冠状動脈のEC（MCECs）を得ることです。心臓から採取した細胞の量は、他の技術を用いて他の組織から採取した細胞よりも少ない場合でも、この技術はまだ賭けを提供しますターの品質。得られた集団における内皮細胞の純度は90％以上です。したがって、この技術は、細胞イメージングとして純粋に単離された内皮細胞に依存するアプリケーションのために理想的です。

<table><tbody><tr><td>BSA</td><td>Sigma</td><td>A3311-10G</td><td></td></tr><tr><td>EDTA</td><td>Fisher</td><td>BP120-500</td><td></td></tr><tr><td>HBSS</td><td>Fisher</td><td>SH3026801</td><td></td></tr><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>H4034-500G</td><td></td></tr><tr><td>Sodium Bicarbonate&amp;nbsp;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>D-(+)-Glucose&amp;nbsp;</td><td>Sigma</td><td>G7021-1KG</td><td></td></tr><tr><td>DynaBeads pan mouse IgG beads&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11035</td><td></td></tr><tr><td>Rat anti-mouse CD31 Antibody</td><td>BD Biosciences</td><td>550274</td><td></td></tr><tr><td>IFCS&amp;nbsp;</td><td>Fisher</td><td>SH30072.04</td><td></td></tr><tr><td>M199</td><td>Fisher</td><td>MT10060-CV</td><td></td></tr><tr><td>Dispase (Neutral Protease)</td><td>Worthington Biochemical Corp./Fisher</td><td>LS02109</td><td></td></tr><tr><td>Collagenase Type II&amp;nbsp;</td><td>Worthington Biochemical Corp./Fisher</td><td>LS004176</td><td></td></tr><tr><td>Glycerol&amp;nbsp;</td><td>Fisher</td><td>BP381-1</td><td></td></tr><tr><td>Igepal</td><td>Sigma</td><td>I3021-50 ml</td><td></td></tr><tr><td>Phosphatase inhibitor cocktail</td><td>Sigma</td><td>P0044-1ml</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma</td><td>P8340-1 ml</td><td></td></tr><tr><td>Heparin&amp;nbsp;</td><td>Sigma</td><td>H3149-100KU</td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>Fisher/Mediatech</td><td>MT35010CV</td><td></td></tr><tr><td>D-Valine</td><td>Sigma</td><td>V1255-5G</td><td></td></tr><tr><td>EGS</td><td>Corning</td><td>356006</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Fisher/Mediatech</td><td>MT-30-002-CI</td><td></td></tr></tbody></table>

isolation of mouse coronary endothelial cells

内皮細胞は、血管の内壁を裏打ちし、血管緊張、血管透過性、および新血管形成の調節に重要な役割を果たしています。内皮細胞の機能障害は、虚血性心疾患を含む多くの心血管疾患の発症および進行に関与しています。冠動脈内皮細胞の機能と特徴付けを調べるために、細胞の単離は、最初のステップであり、それはその後の実験を行うために、高純度と量を必要とします。このプロトコルは、成体マウス冠動脈内皮細胞を単離するための効率的な方法を説明します。マウスの心臓を切開し、小片に刻んだされます。ディスパーゼおよびコラゲナーゼIIを使用して、心臓の消化の後、細胞を洗浄し、抗CD31抗体に結合された磁気ビーズと共にインキュベートしました。内皮細胞とビーズを数回洗浄し、様々な用途で使用する準備ができている、イメージングおよび分子生物学的experime含めていますNTS。効率的な分離は、90％以上の純度で1心あたり約10 4個の細胞が得られます。

マウスの心臓から冠動脈内皮細胞の正常な分離は、典型的には、石畳の形状と90％以上の純度で約10 4個の細胞が得られます。内皮細胞の純粋な集団のために、注意が汚染のない無菌環境を確保するためのプロトコル全体で取られるべきです。細長い細胞または集団内の拡大された細胞の出現は、他の細胞型、平滑筋細胞および線維芽細胞の混入を示し...

Watch this Scientific Journal Video about Isolation of Mouse Coronary Endothelial Cells at JoVE.com

Isolation of Mouse Coronary Endothelial Cells

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

マウスの冠動脈内皮細胞の単離

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

isolation-of-mouse-coronary-endothelial-cells

Research

JoVE Journal

Biology

11.1K ビュー.  University of Arizona, Tucson. This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

ビデオ: Isolation of Mouse Coronary Endothelial Cells

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

ラット心臓の心室自由壁からの心内膜細胞と冠状動脈内皮細胞の分離

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...

発生生物学

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

新生児マウスからの肺血管内皮細胞の単離と培養

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

生物学

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

プライマリ マウス骨格筋の微小血管内皮細胞の分離

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

神経科学

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

マウス抵抗動脈から毛細血管内皮の管の隔離

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Isolation of Primary Mouse Lung Endothelial Cells

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in medical conditions including diabetes, atherosclerosis, sepsis, and acute lung injury. A reliable and reproducible method to isolate pure endothelial cells from mice is needed to investigate the role of endothelial cells in the pathogenesis of these and other conditions. In this protocol, lung microvascular endothelial cells were prepared from 5-7 day old neonatal mouse pups. Lungs are harvested, minced, and enzymatically digested with collagenase I, and released cells are cultured overnight. Endothelial cells are then selected using anti-PECAM1 (CD-31) IgG conjugated to magnetic beads, and cells again are cultured to confluence. A secondary cell selection then occurs with anti-ICAM2 (CD-102) IgG conjugated to magnetic beads to increase the purity of the endothelial cells, and the cells again are cultured to confluence. The entire process takes approximately 7-10 days before the cells can be used for experimentation. This simple protocol yields highly pure (purity &#62;92%) endothelial cells that can be immediately used for in vitro studies, including the studies focused on endothelial cytokine and chemokine production, leukocyte-endothelial interactions, endothelial coagulation pathways, and endothelial permeability. With many knockouts and transgenic mouse lines available, this procedure also lends itself to understanding the function of specific genes expressed by endothelial cells in healthy and pathologic responses to injury, infection, and inflammation.

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

初代マウス肺内皮細胞の単離

Endothelial cells play critical roles in the regulation of vascular tone, immunity, coagulation, and permeability. Endothelial dysfunction occurs in ...

免疫・感染学

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

ネズミ弁内皮細胞の単離

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

マウス大動脈内皮細胞の単離および初代培養

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

マウス冠動脈血管平滑筋細胞の単離

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

マウス初代肝臓正弦波内皮細胞の単離と特性評価

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in the endothelium at the transcriptomic and epigenomic levels, resulting in proatherogenic responses. Recently, single-cell RNA sequencing (scRNAseq) and single-cell Assay for Transposase Accessible Chromatin sequencing (scATACseq) studies were performed to determine the transcriptomic and chromatin accessibility changes at a single-cell resolution using the mouse partial carotid ligation (PCL) model. As endothelial cells (ECs) represent a minor fraction of the total cell populations in the artery wall, a luminal digestion method was used to obtain EC-enriched single-cell preparations. For this study, mice were subjected to PCL surgery to induce d-flow in the left carotid artery (LCA) while using the right carotid artery (RCA) as a control. The carotid arteries were dissected out two days or two weeks post PCL surgery. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single cells or single nuclei were obtained. These single-cell and single-nuclei preparations were subsequently barcoded using a 10x Genomics microfluidic setup. The barcoded single-cells and single-nuclei were then utilized for RNA preparation, library generation, and sequencing on a high-throughput DNA sequencer. Post bioinformatics processing, the scRNAseq and scATACseq datasets identified various cell types from the luminal digestion, primarily consisting of ECs. Smooth muscle cells, fibroblasts, and immune cells were also present. This EC-enrichment method aided in understanding the effect of blood flow on the endothelium, which could have been difficult with the total artery digestion method. The EC-enriched single-cell preparation method can be used to perform single-cell omics studies in EC-knockouts and transgenic mice where the effect of blood flow on these genes has not been studied. Importantly, this technique can be adapted to isolate EC-enriched single cells from human artery explants to perform similar mechanistic studies.

We present a method for isolating endothelial cells and nuclei from the lumen of mouse carotid arteries exposed to stable or disturbed flow conditions to perform single-cell omics experiments.

シングルセルマルチオミクス実験のためのマウス頸動脈内腔からの内皮細胞の単離

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in ...

マウスの冠動脈内皮細胞の単離

要約

さらに動画を探す

この動画の章

ラット心臓の心室自由壁からの心内膜細胞と冠状動脈内皮細胞の分離

新生児マウスからの肺血管内皮細胞の単離と培養

プライマリマウス骨格筋の微小血管内皮細胞の分離

マウス抵抗動脈から毛細血管内皮の管の隔離

初代マウス肺内皮細胞の単離

ネズミ弁内皮細胞の単離

マウス大動脈内皮細胞の単離および初代培養

マウス冠動脈血管平滑筋細胞の単離

マウス初代肝臓正弦波内皮細胞の単離と特性評価

シングルセルマルチオミクス実験のためのマウス頸動脈内腔からの内皮細胞の単離

マウスの冠動脈内皮細胞の単離

要約

さらに動画を探す

この動画の章

ラット心臓の心室自由壁からの心内膜細胞と冠状動脈内皮細胞の分離

新生児マウスからの肺血管内皮細胞の単離と培養

プライマリ マウス骨格筋の微小血管内皮細胞の分離

マウス抵抗動脈から毛細血管内皮の管の隔離

初代マウス肺内皮細胞の単離

ネズミ弁内皮細胞の単離

マウス大動脈内皮細胞の単離および初代培養

マウス冠動脈血管平滑筋細胞の単離

マウス初代肝臓正弦波内皮細胞の単離と特性評価

シングルセルマルチオミクス実験のためのマウス頸動脈内腔からの内皮細胞の単離

プライマリマウス骨格筋の微小血管内皮細胞の分離