Bu araştırma, Amerikan Kalp Birliği hibe 0950118G.to MC-W tarafından kısmen desteklenmiştir.

1. , Anti-PECAM-1 antikor konjuge manyetik boncuk hazırlanması (Dynabeads) <ol><li> PBS içinde% 0,1 BSA BSA eriyene kadar bir vorteks kullanarak 50 ml PBS içinde 50 mg BSA karıştırma hazırlayın. 0.22 mikron şırınga filtre ile filtreleme ile sterilize edin. 4 ° C saklayınız </li><li> TC kaput, 1.5 ml Eppendorf tüp içine yeniden süspanse koyun anti-fare IgG Dynabeads 200 ul transferi ve boncuklar yıkamak: Dağı tüp MPC ve tortu boncuk izin vermek için yaklaşık 1 dakika bekletin. Süpernatant aspire, MPC tüp kaldırmak ve 1 ml steril% 0,1 BSA / PBS boncuklar tekrar süspansiyon haline getirin. Boncuklar tekrar süspansiyon pipet ve aşağı yukarı. </li><li> Toplam dört yıkama, yıkama üç kez daha tekrarlayın. </li><li> MPC&#39;den tüpünü çıkarın ve% 0,1 BSA / PBS 500 ul boncuklar tekrar süspansiyon haline getirin. </li><li> Tüpüne 10 ul sıçan anti-fare PECAM-1 (CD-31) antikor ekleyin. </li><li> Soğuk oda veya oda sıcaklığında 2 saat 4 ° C&#39;de gece Tumble. </li><li> Adım 1.2 dört kez açıklandığı gibi steril% 0,1 BSA / PBS ile boncuk yıkayın. </li><li> MPC&#39;den tüpünü çıkarın ve 200 ul% 0,1 BSA / PBS boncuklar tekrar süspansiyon haline getirin. Mağaza, anti-PECAM-1 antikor konjuge Dynabeads 4 ° C 1 aya kadar. </li></ol> 2. Yenidoğan farelerin Yalıtımlı fare pulmoner endotel hücreleri Doku arıtma protokol geçmeden önce, işlemin IACUC Komitesi onayı gereklidir. <ol><li> Aşağıdakileri hazırlayın: <ul><li> DMEM 1 mg / ml &#39;C / D çözümü, 15 ml. 37 filtre ve sıcak 0.22 mikron şırınga ile Filtre ° C </li><li> 50 ml konik tüp 15 ml DMEM, buz üzerinde mağaza ile </li><li> DMEM% 20 FBS ve 1x penisilin / streptomisin içeren İzolasyon Media (IM) </li><li> Jelatin 2%. 100 ml, 4 gg su, otoklav 15 dakika ve mağaza ° C 2 g sığır cilt jelatin karıştırın Sıcak - 37 ° C kaplama TC şişeler önce sıvılaştığını. </li></ul></li><li> Ketamin (100 mg / kg) ve xylazine (10 mg / kg) bir IP enjeksiyon kullanarak üç 6-8 günlük yavrular anestezisi. Aşağıdaki gibi anestezi etkinliğini kontrol edin ve hayvanların tek tek teşrih: kuruluna Pin yavrular, steril diseksiyon makas ile göğüs bölgesinde cilt,% 70 Etanol ile deri emmek ve kaldırmak. Akciğerler maruz Vergi göğüs kafesi. </li><li> Aseptik tüketim bireysel akciğer lobları, dikkatli olmak, bronşlar ve akciğerlerin etrafında görünür bir bağ dokusu incelemek. Buz DMEM tüm akciğerler birleştirin. Yavrular Euthanize. </li><li> TC kaput, 100 mm TC çanak yer DMEM akciğer lobları kaldırmak ve aşırı DMEM aspire. </li><li> Steril makas kullanarak, ~ 100 kere keserek ince doku kıyma. Enzimatik sindirim için 15 ml sıcak C / D çözümü transfer. Rotator 37 ° C&#39;de 45 dakika inkübe edin. </li><li> TC kaput, tek bir hücre süspansiyonu içine 14 gr kanül bağlı ve çiğnemek kümeleri, en az 12 kere, 20 ml şırınganın içine sindirilmiş doku süspansiyon aspire. </li><li> 70 mikron hücre süzgecinden doku süspansiyonu Pass ve süzgeç sindirim durdurmak için 15 ml IM ile yıkayın. </li><li> Pelet 400x g de 5 dakika santrifüj hücre süspansiyonu. </li><li> 3 ml% 0,1 BSA / PBS aspire süpernatant ve tekrar süspansiyon pelet. </li><li> Hücre süspansiyonu 5 ml yuvarlak alt polistiren tüp transferi ve 22.5 ul anti-PECAM-1 antikor konjuge Dynabeads ekleyin. 12 dakika oda sıcaklığında Tumble. </li><li> Kat 2 ml% 2 jelatin ve aspirat fazla jelatin ile T75 şişesi. TC kaput kaplama öncesi hücreleri yaklaşık 30 dakika içinde kuru jelatin izin ver. </li><li> (Adım 2.10), boncuk inkübasyon tamamlandıktan sonra üç Eppendorf tüpleri içine eşit hücre süspansiyonu bölünmüş ve MPC monte. </li><li> Boncuklar, MPC (~ 1 dak.) Kullanarak tortulaşmış olduğunda, supernatant toplamak MPC&#39;den tüp kaldırmak, boncuk yıkayın ~ 1 ml% 0,1 BSA / PBS, MPC ve sonra yeniden bağlayın. </li><li> Dört kez (toplam 5 yıkar) yıkamak tekrarlayın. </li><li> VascuLife 1 ml EnGS-Mv Yaşam Faktörler Kit ve tüp başına 1x penisilin / streptomisin (VL) ile yeniden süspanse boncuk, iyi karıştırma. </li><li> Bir ön-T75 balon kaplı Plaka ve her bir cam şişe içinde toplam hacim 10 ml VL getirmek. </li><li> Büyüme, 1 tam gün izin medya ertesi gün değiştirin ve sonra her gün medyanın yarısını değiştirmek. Hücreleri (genellikle 3-4 gün sonra kültür)&gt;% 70-80 konfluent veya daha fazla zaman, anti-ICAM-2 Dynabeads sıralanabilir. </li></ol> 3. Hazırlanması, anti-ICAM-2 antikor konjuge Dynabeads <ol><li> Anti-ICAM-2 antikor konjuge Dynabeads konfluent kadar anti-PECAM-1 Dynabeads antikor konjuge ve kültür ile seçilmiş olan hücreler daha fazla saflaştırmak için kullanılır. Bu prosedür, bu anti-fare ICAM-2 (CD-102) antikor, anti-PECAM-1 antikor yerine kullanılır dışında, anti-PECAM-1 antikor konjuge Dynabeads (1, yukarıda) anlatıldığı gibi yapılır. </li><li> Anti-ICAM-2 antikor konjuge Dynabeads ° C için 4 saklanabilir1 aya kadar. </li></ol> 4. Anti-ICAM-2 Dynabeads Fare Akciğer Endotel Hücreleri Sıralama <ol><li> % 2 jelatin ile Coat T75 doku kültürü şişesi. </li><li> Hücreleri ile balon konfluent T75 medya aspire ve 8 ml PBS ile yıkayın. </li><li> Aspire PBS, 2 ml% 0.05 tripsin / EDTA (yaklaşık 5 dakika) hücreleri ayırmak. Ayrılmakta yardım şişeler hafifçe dokunun. </li><li> Hücreleri müstakil, tripsin ve kazıma hücrelerini inhibe kalan yapışık hücreleri ayırmak için 2 ml IM ekleyin. 15 ml tüp Transfer hücre süspansiyonu ve 400x g. az 5 dakika aşağı doğru döndürün </li><li> 2 ml% 0,1 BSA / PBS medya ve tekrar süspansiyon hücre pelletini aspire edin. </li><li> 5 ml polistiren yuvarlak alt tüp transferi ve 10 ul anti-ICAM-2 Dynabeads ekleyin. RT 12 dakika Tumble. </li><li> MPC üzerinde iki adet 1,5 ml Eppendorf tüpleri ve montaj Split hücre süspansiyonu. </li><li> Boncuklar bir dakika süreyle uyulması sonra süpernatant aspire edin. MPC&#39;den tüpler çıkarın ve 1 ml% 0,1 BSA / PBS her tüp tekrar süspansiyon haline getirin. PPK üzerinde tekrar bağlayın. </li><li> Dört kez (toplam 5 yıkar) yıkayın tekrarlayın. </li><li> Her tüpe 1 ml VL ile yeniden süspanse edin ve bir hücre sayımı gerçekleştirmek. </li><li> , T25, termos ve / veya T75 şişesi ortalama 750 bin-1 milyon hücre başına 250-350K hücreleri Plaka hücreleri. </li><li> T25 şişelerde 5 ml, VL ve T75 şişeler 10 ml hacim kazandırın. </li><li> Her gün medya değiştirin. Kültür genellikle 2-3 gün, yaklaşık 80% confluency ulaştığında Hücreler deneyler için kullanılabilir. </li></ol> 5. Temsilcisi Sonuçlar Tipik olarak, 6-7 gün sonra hücrelerin ilk hazırlık, yaklaşık 1,2 elde edebilmek üç 6-8 gün eski yavrular 1.5 x 10 6 pulmoner endotel hücreleri. Hücreler ışık mikroskobu altında tipik &quot;arnavut kaldırımı&quot; morfolojisi ekran ve endotel hücreleri (Şekil 1) için karakteristik olan VE-cadherin (CD144) hücre-hücre kavşaklarda boyama, göstermek. MLECs ifade VE cadherin ve VEGFR2 çoğunluğu FACS analizi (Şekil 2) göstermiştir. Genellikle, ilk MLEC izolasyon sonra iki hafta içinde deneyler için bunları kullanmak. <img src="/files/ftp_upload/2316/2316fig1.jpg" alt="Şekil 1" /> Şekil 1 konfluent MLEC tek tabaka mikroskobik analizi. (A) Işık mikroskobu görüntü endotel hücreleri için tipik kültür hücrelerinin arnavut kaldırımlı morfolojisi gösterir. Birçok hücre perinükleer alanda bulunan Tekdüzen yuvarlak yapılar MLEC izolasyonu için kullanılan manyetik boncuk. (B) Endotel özel VE-kaderin konfokal mikroskobi kullanarak gösterildiği gibi hücre-hücre kavşaklarda lokalize olur. Bar 20μm bir temsilcisidir. <img src="/files/ftp_upload/2316/2316fig2.jpg" alt="Şekil 2" /> Şekil 2, endotel-spesifik belirteçler için spesifik antikorların etiketli kültürlü MLECs FACS analizi: fikoeritrin-konjuge sekonder antikor takip VE kaderin (CD144) veya VEGFR2 olarak suçlanan, izole MLECs endotel kimliğini doğruluyor . Kırmızı çizgi izotip özel kontrol gösterir, yeşil hat gösterir anti-VE-kaderin ya da anti-VEGFR2 özgü IgG.

<ol>
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Structure, function, and mechanisms.</a> Circ Res. 100, 158-173 (2007).</li><li>Fehrenbach, M. L., Cao, G., Williams, J. T., Finklestein, J. M., Delisser, H. 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+of+murine+lung+endothelial+cells.">Isolation of murine lung endothelial cells.</a> Am J Physiol Lung Cell Mol Physiol. 296, L1096-L1103 (2009).</li><li>Miebach, S., Grau, S., Hummel, V., Rieckmann, P., Tonn, J. C., Goldbrunner, R. H. <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+microvascular+endothelial+cells+from+gliomas+of+different+WHO+grades.">Isolation and culture of microvascular endothelial cells from gliomas of different WHO grades.</a> J. Neurooncol. 76, 39-48 (2006).</li><li>Demeule, M., Labelle, M., R&#233;gina, A., Berthelet, F., B&#233;liveau, R. <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+brain,+lung,+and+kidney:+expression+of+the+multidrug+resistance+P-glycoprotein+isoforms.">Isolation of endothelial cells from brain, lung, and kidney: expression of the multidrug resistance P-glycoprotein isoforms.</a> Biochem. Biophys. Res. Commun. 281, 827-834 (2001).</li><li>Kajimoto, K., Hossen, N., Hida, K., Ohga, N., Akita, H., Hyodo, M., Hida, Y., Harashima, H. <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+microvascular+endothelial+cells+from+murine+inguinal+and+epididymal+adipose+tissues.">Isolation and culture of microvascular endothelial cells from murine inguinal and epididymal adipose tissues.</a> J. Immunol. Methods. 357, 43-50 (2010).</li><li>Breviario, F., Caveda, L., Corada, M., Martin-Padura, I., Navarro, P., Golay, J., Introna, M., Gulino, D., Lampugnani, M. 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A., Grammas, P., Giacomelli, F., Wiener, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+heart-derived+endothelial+and+smooth+muscle+cell+cultures:+isolation,+cloning+and+characterization.">Rat heart-derived endothelial and smooth muscle cell cultures: isolation, cloning and characterization.</a> Tissue Cell. 20, 477-492 (1988).</li><li>Petzelbauer, P., Bender, J. R., Wilson, J., Pober, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+dermal+microvascular+endothelial+cell+antigen+expression+and+cytokine+responsiveness+in+situ+and+in+cell+culture.">Heterogeneity of dermal microvascular endothelial cell antigen expression and cytokine responsiveness in situ and in cell culture.</a> J Immunol. 151, 5062-5072 (1993).</li><li>Chrzanowska-Wodnicka, M., Kraus, A. 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Protokolü AUA # 00001206 altında Wisconsin IACUC Komitesi Tıp Fakültesi tarafından belirlenen kurallara ve yönetmeliklere uygun olarak hayvanlar üzerinde deneyler yapıldı.

Mikrovasküler EC vasküler biyoloji birçok alanda yararlı bir model olduğu kanıtlanmış ve yaygın olarak incelenmiştir HUVECs 3 &#39;ünden (örneğin anjiyogenez) incelemek için daha fazla fizyolojik ilgili olduğu düşünülmektedir . Daha önce, bu mikrovasküler EC böbrek, kalp, deri, retina, beyin, gliomlar, yağ dokusu ve aynı zamanda akciğer 2, 4-7, 10, 11 elde edilebilir olduğu bildirilmiştir. Ancak, tutarlı ve güvenilir bir yöntemdir mikrovasküler EC izolasyonu için hala gereklidir. Burada sunulan yöntemle, daha önce yayınlanmış bir protokol 2 bir değişiklik; yavrular yerine yetişkin hayvanların kullandığı en yayınlanan prosedürleri farklıdır. Genç hayvanlardan elde edilen hücrelerin çoğalması potansiyeli yüksek ve dokulardan elde edilen kültürler, hücrelerin daha fazla sayıda verim eğilimi olarak bu izolasyon başarısı için çok önemlidir. Bir hafta eski hayvanların en iyi gibi görünüyor, ancak genç farelerde (4 gün eski) de kullanılabilir. Ayrıca, yavruların daha yüksek veya daha düşük sayıda bir yavru MLECs izole başarılı olduğu gibi, gerektiğinde kullanılan olabilir. Ancak, izolasyon işlemini ölçekleme veya aşağı iken, hücre kaplama yoğunluğu bu da hücre çoğalması için kritik olarak değişmeden kalmalıdır. Ilk anti-PECAM-1 konjuge manyetik boncuklar kullanarak MLECs ve anti-ICAM-2 antikorları daha 2 adım arınma süreci çok daha verimli, daha önce açıklanan tek bir adım arıtma işlemleri daha saf EC kültürler elde. Bu protokol, çok zahmetli bir el teknikleri, degrade santrifüj ve fibroblastlar, kan hücreleri ve düz kas hücreleri gibi atarak kirlenmesine neden olan hücreler için sıralama daha az verimli FACS kullanma ihtiyacı ortadan kaldırır. Anjiyogenik yanıtları, damar geçirgenliği ve lökosit göçüne in vitro analizi, iyileşme yanı sıra sinyal yolları biyokimyasal analiz yara sonucu MLEC nüfus kullanılabilir. Gerekirse, MLECs trans-endotelyal direnci (TER) ölçümleri için immünofloresan veya elektrod fibronektin veya jelatin kaplı lamelleri gibi farklı yüzeylerde kaplama olabilir. Elde edilen sonuçlar, giderek artan sayıda nakavt ve transgenik fare hatları bağlamında özellikle önemlidir in vivo gözlenen fenotipleri doğrudan mukayese edilebilir . In vitro analizi, in vivo gözlenen hatalarının altında yatan hücresel mekanizmaları için bu yöntemi başarılı bir şekilde uygulanması, zaten 12 sunulmuştur .

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Spring scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15032-13</td>
<td>&nbsp;</td>
<tr>
<td>forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11223-20</td>
<td>&nbsp;</td>
<tr>
<td>14G cannula</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>1482516N</td>
<td>&nbsp;</td>
<tr>
<td>20 ml syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309661</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich </td>
<td>A7030-100G</td>
<td>&nbsp;</td>
<tr>
<td>anti-rat IgG Dynabeads</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>110.35</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Particle Concentrator (MPC)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>123-21D</td>
<td>&nbsp;</td>
<tr>
<td>anti-mouse PECAM-1 antibody (CD-31)</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>553369</td>
<td>&nbsp;</td>
<tr>
<td>anti-mouse ICAM-2 antibody (CD-102) </td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>553326</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase/Dispase (C/D)</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>11097113001</td>
<td>100mg/ml stock solution can be prepared and stored at -20&deg;C</td>
</tr>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Cellgro Mediatech</td>
<td>10-013-CV</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Skin Gelatin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#G9391-100G</td>
<td>&nbsp;</td>
<tr>
<td>Vasculife EnGS-Mv complete kit (VL)</td>
<td>&nbsp;</td>
<td>Lifeline Cell Technology</td>
<td>LL0004</td>
<td>&nbsp;</td>
<tr>
<td>0.05% Trypsin/EDTA </td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>25-052-CI</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer 70 &mu;m nylon</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>tissue culture flask 75 cm2</td>
<td>&nbsp;</td>
<td>TPP</td>
<td>90076</td>
<td>&nbsp;</td>		</tbody>
		</table>
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isolation and culture of pulmonary endothelial cells from neonatal mice

Endotel hücreleri, damar biyoloji birçok alanda yararlı bir araştırma modeli sağlar. Ilk izolasyon 1 yılından bu yana, insan umbilikal ven endotel hücreleri (HUVECs) en çok araştırılan endotel hücreleri, böylece kolay elde etmek ve kültürüne uygun görüldüğü gibi, ve. Ancak, anjiyogenez, geçirgenlik ya da diğerleri gibi süreçler üzerinde odaklanan araştırma, mikrovasküler endotel hücreleri (EC) 2. çalışma çok daha önemli fizyolojik model . Ayrıca, nakavt fareler izole edilen EC protein fonksiyon ex vivo analizi için yararlı bir araç sağlar . Çeşitli yaklaşımlar izole etmek ve kültür mikrovasküler farklı kökenli EC tarihten 3-7 bildirilen, ancak saf EC tutarlı izolasyonu ve kültürü hala birçok laboratuvarda büyük bir teknik sorun var. Burada, biz bir adım-adım protokol yalıtma ve kültür fare akciğer endotel hücreleri (MLECs) güvenilir ve nispeten basit bir yöntem sağlar. Bu yaklaşımda, akciğer dokusu 6 elde edilen 8 gün eski yavrular ilk kollajenaz / dispase (C / D) çözümü ile sindirilmiş, parçalar halinde kesilir ve tek hücre süspansiyonu içine mekanik dağınık. MLECS Manyetik Parçacık Yoğunlaştırıcı (PPK) kullanarak Dynabeads konjuge anti-PECAM-1 antikoru ile pozitif seçim kullanarak hücre süspansiyonu arınmış. Konfluent olana kadar saflaştırılmış Böyle hücreleri (TC) yemekleri, jelatin kaplı doku kültürü kültüre. Bu noktada, hücrelerin daha fazla anti-ICAM-2 antikoru ile birleştiğinde Dynabeads kullanarak saflaştırılmış. Bu protokol ile elde MLECs bir arnavut kaldırımlı fenotip, sergi, faz-kontrast ışık mikroskobu olarak görünür ve kendi endotel fenotipi anti-VE-kaderin 8 ve anti-VEGFR2 9 antikorlar ve immunofluorescent VE kaderin boyama FACS analizi kullanılarak teyit edilmiştir. Bizim ellerde, bu iki adımlı bir izolasyon prosedürü, tutarlı ve güvenilir MLECs saf bir nüfus, daha kültürlü olabilir verir. Bu yöntem, araştırmacılar, eleme ve izole MLECs üzerinde gerçekleştirilen in vitro deneylerde sonuçları in vivo çalışmalar doğrudan ilişkilendirmek için transgenik fareler sayıları giderek artan yararlanmak ve bu nedenle in vivo gözlenen vasküler fenotipleri moleküler mekanizmaları ortaya çıkarmak için yardım sağlayacaktır.

Watch this Scientific Journal Video about Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice at JoVE.com

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Burada, fare pulmoner endotel hücrelerin izolasyonu ve kültürü için bir protokol açıklar. Bu yöntem, mekanik ve enzimatik akciğer dokusunda ayrılma yanı sıra anti-PECAM-1 ve çoğunlukla mikrovasküler kökenli saf bir endotel hücre popülasyonu üreten manyetik boncuk, konjuge anti-ICAM-2 antikorları kullanarak 2-aşamalı bir arınma süreci kapsar.

Yenidoğan Fareler Pulmoner Endotel Hücreleri İzolasyon ve Kültür

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

isolation-culture-pulmonary-endothelial-cells-from-neonatal

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

47.0K Görüntüleme.  BloodCenter of Wisconsin. Burada, fare pulmoner endotel hücrelerin izolasyonu ve kültürü için bir protokol açıklar. Bu yöntem, mekanik ve enzimatik akciğer dokusunda ayrılma yanı sıra anti-PECAM-1 ve çoğunlukla mikrovasküler kökenli saf bir endotel hücre popülasyonu üreten manyetik boncuk, konjuge anti-ICAM-2 antikorları kullanarak 2-aşamalı bir arınma süreci kapsar.

Video: Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

İnsan umbilikal ven endotel hücreleri İzolasyon (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Biyoloji

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Kapak Endotel Hücreleri İzolasyon

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

İnsan Cilt Yetişkin Epitel Kök Hücre İzolasyonu ve Kültür

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

İzolasyon ve Kültür Yenidoğan Fare kardiyomiyositlerin

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Embriyonik Ventriküler Endotel Hücre İzolasyonu

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

Yenidoğan fareler ile Pulmoner Arter Düz Kas Hücre İzolasyonu

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

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.

Murin Vana Endotel Hücreleri İzolasyon

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.

İzolasyon ve Fare aort endotel hücreleri İlköğretim Kültür

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

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

İnsan çevresel kanından üretilmesi ve kültür Blood üremesi Endotel Hücreleri

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

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.

Fare Koroner Endotel Hücreleri İzolasyon

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