Name: a cell culture model of resistance arteries
Uploaded: 2017-09-08T12:00:00
Description: Watch this Scientific Journal Video about A Cell Culture Model of Resistance Arteries at JoVE.com

この作品は、各国用の中心によって支えられた、肺、血液研究所 R01088554、T32HL007284、アメリカの心臓協会の交わりを経て 14PRE20420024 を付与します。我々 は特に、聖ジュード細胞イメージング共有研究コア、ランドール ウェイク フィールドと電子顕微鏡画像のリンダ ・ ホーナー、アダム ストラウブについては代表的な蛍光画像彼女の地下鉄 Bagher などを感謝します。原稿プロトコルに関する貴重なフィードバック。

1.、VCCC のめっきセル <ol><li>文化大 225 cm 2 のフラスコ EC と SMC 70-90% 合流までの 37 ° C で滅菌条件の下で。SMC よりより多くの EC が使用されていることを確認します。プライマリ人間 EC と SMC、SMC の 1 大規模なフラスコに EC の通常 3 の大きなフラスコを使用して場合。
	<ol><li>下通路で一次電池を使用して、過去の 10 の通路を使用しないでください。共培養する細胞に基?...

<ol>
	<li>Isakson, B. E., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterocellular+contact+at+the+myoendothelial+junction+influences+gap+junction+organization.">Heterocellular contact at the myoendothelial junction influences gap junction organization.</a> Circ Res. 97 (1), 44-51 (2005).</li><li>Little, T. L., Xia, J., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dye+tracers+define+differential+endothelial+and+smooth+muscle+coupling+patterns+within+the+arteriolar+wall.">Dye tracers define differential endothelial and smooth muscle coupling patterns within the arteriolar wall.</a> Circ Res. 76 (3), 498-504 (1995).</li><li>Heberlein, K. R., Straub, A. C., Isakson, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myoendothelial+junction:+breaking+through+the+matrix?.">The myoendothelial junction: breaking through the matrix?.</a> Microcirculation. 16 (4), 307-322 (2009).</li><li>Straub, A. C., Zeigler, A. C., Isakson, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myoendothelial+junction:+connections+that+deliver+the+message.">The myoendothelial junction: connections that deliver the message.</a> Physiology (Bethesda). 29 (4), 242-249 (2014).</li><li>Isakson, B. E., Ramos, S. I., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++and+inositol+1,4,5-trisphosphate-mediated+signaling+across+the+myoendothelial+junction.">Ca2+ and inositol 1,4,5-trisphosphate-mediated signaling across the myoendothelial junction.</a> Circ Res. 100 (2), 246-254 (2007).</li><li>Dora, K. A., Doyle, M. P., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevation+of+intracellular+calcium+in+smooth+muscle+causes+endothelial+cell+generation+of+NO+in+arterioles.">Elevation of intracellular calcium in smooth muscle causes endothelial cell generation of NO in arterioles.</a> Proc Natl Acad Sci U S A. 94 (12), 6529-6534 (1997).</li><li>Sonkusare, S. K., 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=Elementary+Ca2++signals+through+endothelial+TRPV4+channels+regulate+vascular+function.">Elementary Ca2+ signals through endothelial TRPV4 channels regulate vascular function.</a> Science. 336 (6081), 597-601 (2012).</li><li>Ledoux, 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=Functional+architecture+of+inositol+1,4,5-trisphosphate+signaling+in+restricted+spaces+of+myoendothelial+projections.">Functional architecture of inositol 1,4,5-trisphosphate signaling in restricted spaces of myoendothelial projections.</a> Proc Natl Acad Sci U S A. 105 (28), 9627-9632 (2008).</li><li>Boerman, E. M., Everhart, J. E., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+age+decreases+local+calcium+signaling+in+endothelium+of+mouse+mesenteric+arteries+in+vivo.">Advanced age decreases local calcium signaling in endothelium of mouse mesenteric arteries in vivo.</a> Am J Physiol Heart Circ Physiol. 310 (9), H1091-H1096 (2016).</li><li>Toussaint, F., Charbel, C., Blanchette, A., Ledoux, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMKII+regulates+intracellular+Ca(2)(+)+dynamics+in+native+endothelial+cells.">CaMKII regulates intracellular Ca(2)(+) dynamics in native endothelial cells.</a> Cell Calcium. 58 (3), 275-285 (2015).</li><li>Truskey, G. A. <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+Vascular+Smooth+Muscle+Cell+Co-Culture+Assay+For+High+Throughput+Screening+Assays+For+Discovery+of+Anti-Angiogenesis+Agents+and+Other+Therapeutic+Molecules.">Endothelial Cell Vascular Smooth Muscle Cell Co-Culture Assay For High Throughput Screening Assays For Discovery of Anti-Angiogenesis Agents and Other Therapeutic Molecules.</a> Int J High Throughput Screen. 2010 (1), 171-181 (2010).</li><li>Biwer, L. A., 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=Two+functionally+distinct+pools+of+eNOS+in+endothelium+are+facilitated+by+myoendothelial+junction+lipid+composition.">Two functionally distinct pools of eNOS in endothelium are facilitated by myoendothelial junction lipid composition.</a> Biochim Biophys Acta. 1861 (7), 671-679 (2016).</li><li>Straub, A. C., 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+cell+expression+of+haemoglobin+alpha+regulates+nitric+oxide+signalling.">Endothelial cell expression of haemoglobin alpha regulates nitric oxide signalling.</a> Nature. 491 (7424), 473-477 (2012).</li><li>Isakson, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+expression+of+an+Ins(1,4,5)P3+receptor+at+the+myoendothelial+junction+selectively+regulates+heterocellular+Ca2++communication.">Localized expression of an Ins(1,4,5)P3 receptor at the myoendothelial junction selectively regulates heterocellular Ca2+ communication.</a> J Cell Sci. 121 (Pt 21), 3664-3673 (2008).</li><li>Axel, D. I., 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=Induction+of+cell-rich+and+lipid-rich+plaques+in+a+transfilter+coculture+system+with+human+vascular+cells.">Induction of cell-rich and lipid-rich plaques in a transfilter coculture system with human vascular cells.</a> J Vasc Res. 33 (4), 327-339 (1996).</li><li>Hastings, N. E., Simmers, M. B., McDonald, O. G., Wamhoff, B. R., Blackman, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerosis-prone+hemodynamics+differentially+regulates+endothelial+and+smooth+muscle+cell+phenotypes+and+promotes+pro-inflammatory+priming.">Atherosclerosis-prone hemodynamics differentially regulates endothelial and smooth muscle cell phenotypes and promotes pro-inflammatory priming.</a> Am J Physiol Cell Physiol. 293 (6), C1824-C1833 (2007).</li><li>Herzog, D. P., Dohle, E., Bischoff, I., Kirkpatrick, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+communication+in+a+coculture+system+consisting+of+outgrowth+endothelial+cells+and+primary+osteoblasts.">Cell communication in a coculture system consisting of outgrowth endothelial cells and primary osteoblasts.</a> Biomed Res Int. , 320123 (2014).</li><li>Jacot, J. G., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+injury+induces+vascular+smooth+muscle+cell+proliferation+in+highly+localized+regions+of+a+direct+contact+co-culture+system.">Endothelial injury induces vascular smooth muscle cell proliferation in highly localized regions of a direct contact co-culture system.</a> Cell Biochem Biophys. 52 (1), 37-46 (2008).</li><li>Scott-Drechsel, D., 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=A+new+flow+co-culture+system+for+studying+mechanobiology+effects+of+pulse+flow+waves.">A new flow co-culture system for studying mechanobiology effects of pulse flow waves.</a> Cytotechnology. 64 (6), 649-666 (2012).</li><li>van Buul-Wortelboer, M. F., 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=Reconstitution+of+the+vascular+wall+in+vitro.+A+novel+model+to+study+interactions+between+endothelial+and+smooth+muscle+cells.">Reconstitution of the vascular wall in vitro. A novel model to study interactions between endothelial and smooth muscle cells.</a> Exp Cell Res. 162 (1), 151-158 (1986).</li><li>Wilhelm, I., Fazakas, C., Krizbai, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+the+blood-brain+barrier.">In vitro models of the blood-brain barrier.</a> Acta Neurobiol Exp (Wars). 71 (1), 113-128 (2011).</li><li>Heberlein, K. R., 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=A+novel+mRNA+binding+protein+complex+promotes+localized+plasminogen+activator+inhibitor-1+accumulation+at+the+myoendothelial+junction.">A novel mRNA binding protein complex promotes localized plasminogen activator inhibitor-1 accumulation at the myoendothelial junction.</a> Arterioscler Thromb Vasc Biol. 32 (5), 1271-1279 (2012).</li><li>Straub, A. C., 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=Compartmentalized+connexin+43+s-nitrosylation/denitrosylation+regulates+heterocellular+communication+in+the+vessel+wall.">Compartmentalized connexin 43 s-nitrosylation/denitrosylation regulates heterocellular communication in the vessel wall.</a> Arterioscler Thromb Vasc Biol. 31 (2), 399-407 (2011).</li><li>Cucina, A., 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=Vascular+endothelial+growth+factor+increases+the+migration+and+proliferation+of+smooth+muscle+cells+through+the+mediation+of+growth+factors+released+by+endothelial+cells.">Vascular endothelial growth factor increases the migration and proliferation of smooth muscle cells through the mediation of growth factors released by endothelial cells.</a> J Surg Res. 109 (1), 16-23 (2003).</li><li>Heberlein, K. R., 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=Plasminogen+activator+inhibitor-1+regulates+myoendothelial+junction+formation.">Plasminogen activator inhibitor-1 regulates myoendothelial junction formation.</a> Circ Res. 106 (6), 1092-1102 (2010).</li><li>Isakson, B. E., Best, A. K., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+protein+on+actin+bridges+between+endothelium+and+smooth+muscle+in+arterioles+demonstrates+heterogeneous+connexin+expression+and+phosphorylation.">Incidence of protein on actin bridges between endothelium and smooth muscle in arterioles demonstrates heterogeneous connexin expression and phosphorylation.</a> Am J Physiol Heart Circ Physiol. 294 (6), H2898-H2904 (2008).</li><li>Biwer, L. A., Isakson, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum-mediated+signalling+in+cellular+microdomains.">Endoplasmic reticulum-mediated signalling in cellular microdomains.</a> Acta Physiol (Oxf). 219 (1), 162-175 (2017).</li></ol>

VCCC は MEJsの in vitro、さたの面で利点の数が特定のアプリケーションのため、VCCC を利用できるかどうかを決定する際の議論のポイントがあります。たとえば、異なる種類の細胞をこのモデルで使用される場合は、最適化するそれ必要があります。ひと初代細胞を用いてウシ大動脈24、または野生型やノックアウト マウスから細胞を分離し、VCCC1,</...

小径抵抗血管の薄い内弾性板 (IEL) は、内皮細胞 (EC) と平滑筋細胞 (SMC) を分離します。セルはこの弾性マトリックスの穴を介して投影し、ギャップ結合チャネル1,2経由でダイレクトに細胞質接続できます。このユニークな構造は、myoendothelial のジャンクション (MEJ) と呼ばれます。MEJ です小 (血管床によって、0.5 μ m x 約 0.5 μ m)、細胞突起主に内皮細胞から成る3,4同様の平滑筋から発生させてよい。調査官の数はシグナル伝達 MEJ、内皮細胞と平滑筋5 間双方向のシグナル化を促進するため特に重要な場所、それはレンダリングに選択的に発生するネットワークの巨大な複雑さを示しています。 ,6,7,8,9,10。
ただし、そのまま動脈、MEJ のシグナル伝達経路の機構的郭清はむずかしい。MEJ から細胞の投影こそ体内MEJ 血管壁から分離することは現在はないです。このため、VCCC モデル1が開発されました。重要なは、VCCC 複製生理的血管内皮形態11 ・頂間の信号の偏波とセル12MEJ 部分。アルファのヘモグロビンは、血管内皮細胞、MEJ に偏光の発見を容易にするこのユニークなモデルだった。これはアルファのヘモグロビン13を表現しない従来培養内皮細胞とは対照的です。微小血管内皮細胞に興味がある研究者は、特に小径抵抗血管で発生するシグナル伝達経路を分析することが目的の場合、VCCC で培養した血管内皮細胞を使用するより適切な場合があります。
2 つ共培養細胞型に小径孔 (直径 0.4 μ m) を持つフィルターを含む頑丈なプラスチック製の挿入を使用すると、セルで層間の移行できなくなります。それはin vivoよりはるかに時間がまだ MEJs、蛋白質のローカリゼーション、シグナルの1二次メッセンジャーなどの生体内の特性の多くを複製セル間の 10 μ m 間隔の結果します。,14します。 さらに、、VCCC セル アゴニストまたは特定の細胞内コンパートメントに拮抗薬の追加を介して信号の種類に固有のターゲットにできます。たとえば、カルシウムをキレート化するためこれら AM で EC を読み込みとフェニレフリン14SMC を刺激します。これにより、共培養モデル15,16,17,18,19,20,21の他の記述と対照をなしてmRNA および蛋白質、分数を含む、異なる MEJ フィルター孔内の異なる画分を分離することについて説明します。この手法、VCCC を添加できます蛋白質活動12蛋白質のリン酸化の12,23, や転写22mRNA の局在の変化の具体的な調査。異なる立体構造11,18,242 つの細胞の種類の培養が可能だが、底部には、フィルターと SMC の上に EC のめっきが、この資料に記載されます。

<table>
	<tbody>
		<tr>
			<td>24mm diameter, 0.4&#181;m Transwell Polyester membrane filter insert</td>
			<td>Corning</td>
			<td>3450</td>
			<td>This is one 6 well plate of inserts</td>
		</tr>
		<tr>
			<td>225cm2 flask</td>
			<td>Corning</td>
			<td>431082</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plates (without filter inserts)</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary human coronary artery endothelial cells</td>
			<td>Lonza</td>
			<td>CC-2585</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary human coronary artery smooth muscle cells</td>
			<td>Lonza</td>
			<td>CC-2583</td>
			<td></td>
		</tr>
		<tr>
			<td>EBM-2 EC Basal media</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr>
			<td>EC growth factors (EGM2 MV Single quots)</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td>Add to basal media before use</td>
		</tr>
		<tr>
			<td>SmBM SMC basal media</td>
			<td>Lonza</td>
			<td>CC-3181</td>
			<td></td>
		</tr>
		<tr>
			<td>SMC growth factors (SmGM-2 SingleQuot)</td>
			<td>Lonza</td>
			<td>CC-4149</td>
			<td>Add to basal media before use</td>
		</tr>
		<tr>
			<td>0.5% Trypsin-EDTA 10X</td>
			<td>Gibco</td>
			<td>15400-054</td>
			<td>Dilute to 2X with sterile dPBS</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>VWR</td>
			<td>15170-208</td>
			<td></td>
		</tr>
		<tr>
			<td>large petri dishes for SMC Plating (150mm)</td>
			<td>Corning</td>
			<td>353025</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine gelatin</td>
			<td>Sigma</td>
			<td>G-9382</td>
			<td></td>
		</tr>
		<tr>
			<td>Human fibronectin</td>
			<td>Corning</td>
			<td>356008</td>
			<td>5&#181;g/ml of fibronectin and store at -20</td>
		</tr>
		<tr>
			<td>10X Hanks&#39; Buffered salt solution (HBSS)</td>
			<td>Gibco</td>
			<td>14065-056</td>
			<td>Dilute to 1x with sterile water</td>
		</tr>
		<tr>
			<td>10X Dulbecco&#39;s Phosphate buffered saline (dPBS)</td>
			<td>Gibco</td>
			<td>14200-075</td>
			<td>Dilute to 1x with sterile water</td>
		</tr>
		<tr>
			<td>Disposable Curved Scalpel (30mm cutting edge) with handle</td>
			<td>Amazon</td>
			<td>MDS15210</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher</td>
			<td>17-467-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lifter</td>
			<td>Corning</td>
			<td>3008</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>BD Falcon</td>
			<td>353085</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL conical tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>10mm petri dishes</td>
			<td>Falcon</td>
			<td>35-1008</td>
			<td></td>
		</tr>
		<tr>
			<td>Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA lysis reagent</td>
			<td>Qiagen</td>
			<td>79306</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td>Make 4% solution with dPBS</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Fisher</td>
			<td>04-355-305</td>
			<td></td>
		</tr>
		<tr>
			<td>Cavicide disinfectant</td>
			<td>Fisher</td>
			<td>131000</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA protein lysis buffer</td>
			<td>Sigma</td>
			<td>R0278</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonic dismembranator</td>
			<td>Fisher</td>
			<td>Model 50</td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS for harvesting EC/SMC and immunocytochemistry</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>does not need to be sterile</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Embedding/Sectioning/Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Fisher</td>
			<td>P31-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated tissue processer</td>
			<td>Excelsior</td>
			<td>ES</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding station + cold plate</td>
			<td>Leica</td>
			<td>HistoCore Arcadia</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Tek Accu-Edge Microtome blade</td>
			<td>VWR</td>
			<td>25608-961 or 25608-964</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Thermo Fisher</td>
			<td>22-230-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Thermo Fisher</td>
			<td>12-548-5E</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold mounting medium</td>
			<td>Thermo Fisher</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Other Solutions</td>
			<td></td>
			<td></td>
			<td>**Perform all steps in sterile conditions if using the solution on live cells</td>
		</tr>
		<tr>
			<td>Sterile dPBS</td>
			<td></td>
			<td></td>
			<td>Autoclave water to sterilize. Using the sterile water make DPBS (450mL sterile water: 50mL 10X DPBS), mix and filter sterilize (0.2&#181;m filter). 
			Final concentration: 1X</td>
		</tr>
		<tr>
			<td>sterile HBSS</td>
			<td></td>
			<td></td>
			<td>Autoclave water to sterilize. Using the sterile water make HBSS (450mL sterile water: 50mL 10X HBSS), mix and filter sterilize (0.2&#181;m filter). 
			Final concentration: 1X</td>
		</tr>
		<tr>
			<td>Fibronectin stock solution</td>
			<td></td>
			<td></td>
			<td>Use 50mL of sterile water to fibronectin to make stock of 1mL aliquots, then freeze until needed 
			Final concentration: 100&#181;g/mL</td>
		</tr>
		<tr>
			<td>Fibronectin to coat SMC side of VCCC</td>
			<td></td>
			<td></td>
			<td>thaw one aliquot and add 1mL of fibronectin (100&#181;g/mL) to 19mLs of 1X HBSS to reach a final concentration of 5&#181;g/mL. 
			Final concentration: 5&#181;g/mL</td>
		</tr>
		<tr>
			<td>2x Trypsin EDTA solution</td>
			<td></td>
			<td></td>
			<td>Dilute the 10X 0.5% Trypsin-EDTA to 2X with 1X dPBS (40mL DPBS: 10mL 10X Trypsin) . 
			Final concentration: 2X</td>
		</tr>
		<tr>
			<td>Blocking/antibody solution</td>
			<td></td>
			<td></td>
			<td>(9mL 1X PBS, 25mL Triton X100, 50mg bovine serum albumin, 500mL normal serum)</td>
		</tr>
		<tr>
			<td>Bovine gelatin to coat EC VCCC</td>
			<td></td>
			<td></td>
			<td>Mix 0.5g bovine gelatin into 100mL distilled water. Autoclave prior to first use and store at room temperature. Keep in sterile conditions. 
			Final concentration: 0.50%</td>
		</tr>
		<tr>
			<td>1X PBS for harvesting cells</td>
			<td></td>
			<td></td>
			<td>does not need to be sterile</td>
		</tr>
	</tbody>
</table>

a cell culture model of resistance arteries

Myoendothelial ジャンクション (MEJ) 小径抵抗血管でユニークなシグナリングの創製は、血管緊張や血圧を制御することができますシグナリング プロセス特定の蛋白質の局在を展示します。内皮細胞や平滑筋細胞からの投影は、その小型のためサイズ (〜 1 μ m2の面積平均)、上、MEJ と単独で勉強することは困難です。ただし、我々 は MEJ 形成体外、内皮細胞の極性形成とシグナル伝達タンパク質と抵抗の血管壁内のプロセスの郭清では、血管細胞共培養 (VCCC) と呼ばれる細胞培養モデルを開発しました。動脈。VCCC 多数のアプリケーションには、異なる種類の細胞に合わせて適応することができます。モデルは、0.4 μ m 孔体外でMEJs することができますフォームをフィルターの両側に成長して 2 種類の細胞で構成されています。ここでセルのめっきと内皮細胞の分離を介して VCCC を作成する方法について述べる MEJ、および蛋白質の隔離や活動のため使用することができます平滑筋分数の試金。埋め込み、および免疫蛍光分析用断面、そのまま細胞層でフィルターを固定できます。重要なは、このモデルからの発見の多くは生理学的な関連性を強調し、そのまま抵抗動脈を使用して確認されています。

VCCC 用フィルター挿入が小、0.4 μ m 穴。図 1 a、VCCC フィルター挿入の正面ビューは、毛穴、MEJ が生体外で形成できますを示しています。回路図は、一日前に、内皮細胞を反対側 (図 1 b) めっきフィルターの下側にメッキの平滑筋細胞を示しています。セルはメッキ後、EC、MEJ、および SMC の成分は分離の 3 日後?...

Watch this Scientific Journal Video about A Cell Culture Model of Resistance Arteries at JoVE.com

A Cell Culture Model of Resistance Arteries

抵抗血管の細胞培養モデルを説明することで、シグナル伝達経路の内皮細胞、平滑筋や内皮細胞と平滑筋 (myoendothelial ジャンクション) 間の解剖を可能にします。アゴニストまたは蛋白質の隔離、電子顕微鏡、蛍光抗体の選択的なアプリケーションは、この細胞培養モデルを使用して利用できます。

抵抗血管の細胞培養モデル

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

Research

JoVE Journal

Medicine

A Mouse Model of in Utero Transplantation

A Mouse Model of in Utero Transplantation

のマウスモデル子宮内で移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

口腔扁平上皮癌の同所性マウスモデルにおける腫瘍細胞の浸潤の多光子イメージング

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

細胞培養のための人間の削減乳房形成や乳房切除組織の処理

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

マウス精密切り出肺スライスを用いた低酸素肺血管収縮のビデオモルフォメトリクス解析

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

圧力筋運動記録法を用いた抵抗腸間膜動脈における筋原性応答および血管作用を評価します

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

新生児母子分離のマウスモデルにおけるPerigenital感度と前立腺肥満細胞の活性化の評価

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

腫瘍細胞移動の研究および抗浸潤性薬物の患者特異的効果の研究のためのヒト神経膠芽腫の器官型スライス培養モデル

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

犬の動脈と静脈からの初代内皮細胞の単離および培養

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

分離抵抗性ラットの動脈のビデオ顕微鏡を用いた血管制御機構の評価

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

筋肉の不均衡: テストと競技人口の機能的な偏心ハムスト リングの筋力トレーニング

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...