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

Bu çalışma Sağlık (A. Makino için HL115578) National Institutes of gelen hibe ile desteklenmiştir.

fareler üzerinde araştırma Arizona Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanmış ve Ulusal Sağlık Enstitüsü (NIH) kurallarına göre gerçekleştirildi. Not: Bölümler 1, 2 ve 3 kurulum olarak Deneyden bir gün önce yapılmalıdır. 1. hazırlanması Çözümler <ol><li> CD31 tamponu (50 mi) <ol><li> 50 ml&#39;lik bir tüpe 50 mi 1 x fosfat tamponlu salin (PBS) ekleyin. 0.05 g Sığır Serum Albümini (BSA) ve 0.037 g etilendiamin tetraasetik asit (EDTA) tartılır ve bir 50 ml tüp ekleme toz eriyene kadar 1 saat boyunca döndürmek için tüp koydu. 7.4 pH ayarlama yeni bir 50 ml tüp içine 0.2 um&#39;lik bir filtreden filtre ve kullanılana kadar 4 ° C&#39;de muhafaza edin. </li></ol></li><li> HEPES Hank Dengeli Tuz Çözeltisi (HBSS) (500 mi) <ol><li> HBSS çözeltisi içinde 10 mM HEPES yapmak ve iyice karıştırın HEPES 1,1915 g ekleyin. pH ölçümü ve 7.4 ayarlamak. 4 filtre ve mağaza˚C. </li></ol></li><li> 1x Krebs çözeltisi (1 L), <ol><li> 1 L&#39;lik bir şişeye damıtılmış su çift 10 x Krebs çözeltisi [Tablo 1], 100 ml ilave edilir. 2.1 gr sodyum bikarbonat ve 2 g D-glikoz tartılır ve şişeye ilave edin. Karıştırın ve 1 L. toplam ses seviyesini ayarlamak </li><li> Kaput içinde 30 dakika ve filtre solüsyonu% 95 O 2/5% CO2 gazı ile kabarcık çözeltisi. 4 C&#39;de muhafaza ve deney sırasında buz üzerinde tutmak. </li></ol></li></ol> 2. Manyetik Boncuk hazırlanması <ol><li> Her 1.5 ml tüp (örnek başına bir boru) 1 ml CD31 tampon ekleyin. Koyun anti-fare IgG boncuk karışımı ve her numune [Tablo 1], uygun miktar ekleyin. kuvvetlice 30 kez tüpleri çalkalayın ve çalkalama 1 dakika süreyle manyetik plaka koyun. plaka üzerinde iken tüpler açın ve bir kovaya çözümler dökümü. </li><li> Her tüpe 1 mi CD31 tampon ekleyin. 2 ul sıçan anti-fare CD31 anti-eklemeHer tüpe gövde ve 4 ºC de tüpler O / N çevirin. </li></ol> 3. Otoklavlama <ol><li> Tüm cerrahi aletler ve pipet uçları otoklavlayın. 4 farklı çaplarda ipuçları vardır, böylece dar olan en geniş dan, pipet uçları uçlarını kesin. İpuçları için yaklaşık çapları 3 mm, 2.5 mm, 2 mm, sırasıyla 1.5 mm&#39;dir. ipuçları kesin ve 121 ºC sıcaklıkta sterilizasyon 30 dakika ile bir gün önce otoklav. Not: Bölüm 4-11, deney günü yapılmalıdır. </li></ol> 4. hazırlanması Çözümler <ol><li> M199 2% Demir-Müstahkem Buzağı Serumu (IFCS) içeren yıkama tamponu hazırlayın. görüntüleme için, 30 ml / fare hazırlamak. </li><li> enzim çözeltisi (10 mi / numune) hazırlayın. 50 ml&#39;lik bir tüp içine 0.6 birim / dispaz ml ve 1 mg / kollajenaz tip II ml tartılır. M199 ekleyin ve 15 dakika boyunca 4 ° C&#39;de tüp döndürün. insid yeni 50 ml&#39;lik bir tüp içine 0.2 um&#39;lik bir filtreden filtree kaput ve kullanılıncaya kadar 4 ° C&#39;de tutun. </li><li> Heparin ile HBSS / HEPES hazırlayın. 15 ml&#39;lik bir tüp içinde 10 mM HEPES içeren HBSS, 10 ml ekleyin. tüpe Heparin 100 ul ekle ve kullanılana kadar 4 ° C&#39;de tutun. </li></ol> 5. Yıkama Boncuk <ol><li> 4 ° C&#39;de rotator gelen boncuklar tüpleri dışarı atın ve kaput getirmek. Manyetik plaka tüpler koyun ve 1 dakika boyunca çalkalanır. çözelti dökümü ve her tüpe 1 mL CD31 tampon eklemek. </li><li> Manyetik plaka dışında tüplerini alın ve kuvvetlice 30 kez çalkalayın. Manyetik plaka tüpler geri koymak ve 3 yıkar toplam tekrarlayın. tamamlanmasından sonra, 4C geri tüpleri koymak ve kullanılana kadar dönen tutmak. </li></ol> 6. Diseksiyon <ol><li> Kan pıhtılaşmasını önlemek için, heparin 0.1 ml intraperitonal fareler enjekte edilir. Daha sonra pentobarbital 0.01 ml intraperitoneal kullanarak fareler uyutmak, 10 dakika bekleyin. Fare duygu olup olmadığını kontrol edinayak kısma ağrı. </li><li> kafa ile bir polistiren gemide fare yukarı bakacak şekilde yerleştirin ve iğneler ile silah tutun. boğazına kadar üst torakal alanda deriyi kesme forseps ve sert kesme makas kullanın. </li><li> diyaframın hemen üstünde göğüs kafesinin ortasında bir kesim olun. kalp ve akciğerler açığa bir üçgen yapmak için boğaz doğru yanlara ve yukarıya kemik kesti. diyaframın hemen üstünde aorta kesin. </li><li> çekerek olmadan forseps ile timus tutun ve birlikte akciğer ve kalp kaldırmak için dikkatlice herhangi bir bağ dokusu kesmek için makas kullanın. 1 × Krebs bir beher yer dokular, forseps ile sallamak ve 1 × Krebs ile 6 cm çanak aktarın. </li><li> makas ve forseps kullanarak akciğer lobları çıkarın. aort içine 20 G steril kateter yerleştirin. HBSS / Heparin ile koroner dolaşım sisteminde kan yıkayın. , Ventrikül küçük bir kesim yapmak kalbi sallamak ve M199 bir örnek tüpüne transfer. buz üzerinde tüpler tutun. </li></ol><p<html> class = &quot;jove_title&quot;&gt; 7. Doku sindirimi <ol><li> Enzim çözeltisi, 10 ml dolu 6 cm çanak kalp transfer ve makas kullanılarak küçük parçalar halinde kıyma. 15 dakika boyunca 37 ° C kuluçka makinesi içinde yemekler yerleştirin. Doku kolay geçmesine izin vermek için bir kesik ucuna bir ucu aşağı 30 kez yukarı pipetleme tarafından sindirilmiş malzemeleri çalkalayın. </li><li> Başka bir 15 dakika süreyle 37 ºC inkübatör geri koyun. Ajitasyon üç kez tekrarlayın, büyük açıklıklar gelen pipet uçları kullanarak her zaman dar ve her arasında 15 dakika inkübasyon için. </li><li> Son 15 dakika inkübasyondan sonra, dışarı ve kaputun içine numune almak. Pipetleyin çözüm yukarı ve pipet ucu dar açılması ile yeni bir 50 ml tüp içine 40 mikron hücre süzgecinden geçmesi kullanarak aşağı 30 kere. </li><li> 10 ml&#39;lik pipet kullanarak 10 ml yıkama tamponu ile çanak yıkayın ve hücre süzgecinden üzerine aktarın. Oda sıcaklığında 10 dakika boyunca 400 g&#39;de santrifüj ve spin 50 ml tüpler koyun. </li></ol>Boncuk ile itle &quot;&gt; 8. Yıkama ve Konjugasyon <ol><li> pelet bozmadan süpernatantı. iki kez 5 ml yıkama tampon maddesi ile hücreleri yıkayın. </li><li> Daha önce biri bir anda bir tüp boncuk ve çalışma ile 1.5 ml tüpler hazırlandı alın. Manyetik plaka bir 1.5 ml tüp koymak 3 kez sallayın ve açın. </li><li> Son santrifüj sonra, pelet bozmadan mümkün olduğunca süpernatant kaldırmak. kuvvetlice iterek hücre kümeleri ilişkisini. </li><li> 50 ml&#39;lik bir tüpe 1 mL yıkama tamponu ilave edin ve 1 ml pipetle hücreler karıştırın. 1.5 ml tüp solüsyonun dökümü ve boncuk 50 ml&#39;lik bir tüp hücre süspansiyonu 1 ml. boruları 30 kez Flick, daha sonra 30 dakika boyunca 4 ° C&#39;de döner. </li></ol> 9. Hücre Kültürü / Görüntüleme için hazırlanıyor <ol><li> hücreleri kaplama için bir oda hazırlayın. kaplamak için de yüzey bölmeye jelatin çözeltisi (w / v% 5, 300 ul) ilave edin ve 37 ° C&#39;de 30 dakika inkübe edilir. İnkübasyon, rem sonrasıove jelatin ve kuru yüzey. </li><li> Dönme 30 dakika sonra boncuk / hücreleri ile tüpler dışarı atın ve kaput içinde manyetik plaka koymak. 2 dk, dökümü için çalkalayın ve 1 ml yıkama tampon ekleyin. </li><li> plaka üzerinden tüpleri almak, şiddetle 30 kez sonra fiske 30 kez çalkalayın. Manyetik plaka onları geri koymak ve 1 dakika boyunca çalkalanır. 5 yıkama toplam 4 kez tekrarlayın, fakat 1 dakika plaka üzerinde çalkalayarak. </li><li> Son yıkamadan sonra, bir tüp çıkar ve bir anda her tüp biri üzerinde çalışmak. Daha sonra 30 kez fiske 30 kez çalkalayın. Manyetik plaka üzerine koyun ve 1 dakika boyunca çalkalanır. Damper ve (37 ºC&#39;de önceden ısıtılmış) 1 ml% 20 IFCS AK medya ekleyebilirsiniz. </li><li> kuvvetlice 30 kez daha sonra 30 kez fiske sallamak. 500 ul pipetlemeyin ayarlayın ve pipetlemeyin yukarı ve 10 kez aşağı. Her bölmeye 500 ul aktarın. </li><li> 37 ºC inkübatör koymak ve / N O bırakın. Bir sonraki gün,% 10 FBS AT ortam hücreleri yıkayın. </li></ol> Western Blot (WB) Örnekleri 10. hazırlanması <ol><li> Takdönme 1 saat sonra boncuklar / hücreleri ile tüpler dışarı e. Manyetik plaka koyun ve 2 dakika süreyle çalkalayın. Süpernatantı dökümü ve 1 ml soğuk HBSS ekleyin. kuvvetlice 30 kez daha sonra 30 kez fiske sallamak. 3 yıkama için iki kez tekrarlayın, ancak 1 dakika plaka üzerinde çalkalayarak. </li><li> Son yıkamadan sonra, manyetik plaka üzerine tüpleri koymak ve 1 dakika boyunca çalkalanır. Pipet kullanarak tamponu çıkarın ve bir yöntemi daha önce 11,12 açıklanan kullanarak WB için hücrelerin lize. Toplanan lizat genellikle yaklaşık 30 ug protein içerir. </li></ol> 11. Görüntüleme <ol><li> Bir bölme için 250 ul nihai hacimde, medya hücreleri 3 kez yıkayın. hücrelere Dil-acLDL 1.5 ul ekle. Bu noktadan sonra, karanlıkta hücreleri tutun. </li><li> 37 ° C&#39;de 3.5 saat süreyle inkübe edin. Hoechst 1.5 ul ekleyin karıştırın ve PBS ile 37 ˚C.Wash hücre, 30 dakika boyunca inkübe ve oda sıcaklığında 20 dakika süreyle% 4&#39;lük paraformaldehit (PFA) ile tespit edin. </li><li> 30 Mİ-PBS BSA PBS ile iki kez yıkanır ve% 5 ile blokeOda sıcaklığında n. % 1 BSA-PBS ile iki kez yıkanır. ml 2 ug / bir konsantrasyona kadar% 1 BSA-PBS ile BS-lektin FITC seyreltilir ve her bir bölmeye 300 ul ekle. </li><li> 30 dakika boyunca oda sıcaklığında orbital bir karıştırıcı üzerinde odasına koyun. PBS ile 3 kez yıkayın. </li><li> FITC (Lektin boyanma, EC işaretleyici, uyarma / emisyon 488 nm / 519 nm), DAPI (Hoechst, 358 nm&#39;de bir nükleer boyanma, uyarma / emisyon / 30 ms 200 msn pozlama süresi ile floresan mikroskop altında hücrelerin görselleştirmek 461 nm) ve 557 nm / 576 nm) TRITC (acLDL boyaması, EC işaretleyici, uyarma / emisyon 300 milisaniye. 20X objektif lens kullanarak görüntüleri elde edin. </li></ol>

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The authors have nothing to disclose.

Araştırmada en yaygın olarak kullanılan endotel hücreleri nedeniyle rahatlık ve kültür kolaylığı, insan göbek damarı endotel hücreleri (HUVECler) vardır. Bununla birlikte, bir çok araştırma diğer özel organlar 10 arasında endotel hücrelerinin izolasyonu ile elde edilen fizyolojik modelin varlığını gerektirir. Endotel hücreleri, kalp dokusu hücrelerinin çok küçük bir popülasyonu oluşturan; onların izolasyon ve kültür çok zor olabilir kanıtlayabilirim. <p class="jove_c...

Çeşitli kardiyovasküler hastalıklar ve metabolik bozukluklar fare modellerinde hastalarda bulunan benzer fizyolojik ve moleküler değişiklik taşımaktadır. Ayrıca, farelerin genetik değişim bizi hastalıkların gelişimi ve ilerlemesinde belirli genlerin patojenik rolünü araştırmak için olanak sağlayan güçlü bir araçtır. Günümüzde, hücre tipi-özgü gen in-tak ya -kasa farenin kolayca farenin gerçek genetik olarak tadil edilmiş olup olmadığını belirlemek için ilk adımdır spesifik hücre tipleri çok laboratuar ve mRNA ölçümü ve protein seviyeleri üretilir. Endotel endotel hücreleri (EC) hatları, iç damar duvarı ince bir tek tabakadır. Endotel hücreleri, vasküler tansiyon, damar geçirgenliğini ve yeni damar oluşumu 1,2 düzenlenmesinde önemli bir rol oynamaktadır. Endotel disfonksiyonu, birçok patolojik durumlarda ve endotel fonksiyonu değişiklikleri bir işaretidir çeşitli araç yol açabilir olduğunukardiyovasküler hastalıklar 3,4. Fizyolojik ve patofizyolojik koşullar altında endotel hücrelerinin işlevini incelemek böylece önemlidir. Orada ECS 5-8 izole etmek çeşitli yolları vardır ve izolasyon yöntemi doku ve türlerin kullanılacak olan bağlı optimize edilmiş olması gerekir. Farklı dokulardan EC yüzey belirteçleri ile ilgili ve protein ekspresyonu 9 heterojenlik ileri düzey bir olmasıdır. endotel hücrelerinin Başarılı izolasyonu genellikle zor olabilir ve eğitim ve uygulama bir dereceye gerektirir. elde kez, bu protokolde öngörülen izolasyon yöntemi istikrarlı ve verimli olduğunu kanıtlamaktadır. Bu yöntemin genel amacı yüksek kalite ve miktar fare koroner EC (MCECs) elde etmektir. Bir kalp toplanan hücrelerin miktarı diğer teknikleri kullanarak, diğer dokulardan elde hücrelerden daha az olsa da, bu teknik yine de bahsi sağlarter kalitesi. Elde edilen popülasyonda endotel hücrelerinin saflığı% 90 daha büyüktür. Bu nedenle, bu teknik, hücre görüntüleme gibi tamamen izole edilmiş endotel hücreleri kullanan uygulamalar için ideal olacaktır.

<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

Endotel hücreleri, kan damarlarının iç çeperini kaplamak ve damar tonu, vasküler geçirgenlik ve yeni damar oluşumu düzenlenmesinde önemli bir rol oynamaktadır. Endotel hücre fonksiyon bozukluğu, iskemik kalp hastalığı da dahil olmak üzere birçok kardiyovasküler hastalıkların gelişimi ve ilerlemesinde rol oynar. fonksiyonu ve koroner endotel hücrelerinin karakterizasyonu incelemek için, hücre izolasyonu ilk adımdır ve daha sonraki deneyler yüksek saflıkta ve miktarını gerektirir. Bu protokol, yetişkin fare koroner endotel hücrelerini izole etmek için etkili bir yöntem tarif eder. Fare kalp disseke ve küçük parçalar halinde kıyılmış. dispaz ve kolajenaz II kullanılarak kalbin sindirimden sonra hücreler yıkanır ve anti-CD31 antikoru ile konjuge edilmiş, manyetik boncuklar ile inkübe edildi. endotel hücreleri ile boncuk yıkanmış birkaç kez ve görüntüleme ve moleküler biyoloji experime dahil olmak üzere çeşitli uygulamalarda kullanıma hazırNTS. Verimli izolasyon% 90&#39;ın üzerinde saflıkta tek yürek başına yaklaşık 10 4 hücre verir.

Bir fare kalp koroner endotel hücrelerinin başarılı izolasyonu tipik bir Arnavut kaldırımı şekli ile% 90&#39;ın üzerinde saflıkta yaklaşık 10 4 hücre verir. endotel hücreleri saf nüfus için, dikkatli her türlü kirlilikten arındırılmış steril bir ortam sağlamak için protokol boyunca alınmalıdır. uzatılmış hücreler ya da nüfus içinde büyütülmüş hücrelerden görünümü, diğer hücre tipleri, yumuşak kas hücreleri ve fibroblastlar ile k...

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.

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 ...

isolation-of-mouse-coronary-endothelial-cells

Research

JoVE Journal

Biology

11.1K Görüntüleme.  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.

Video: 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.

Endokardiyal ve Koroner Endotel Hücrelerinin Sıçan Kalbinin Ventriküler Serbest Duvarından İzolasyon

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

Gelişim Biyolojisi

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.

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 ...

Biyoloji

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;.

Birincil fare iskelet kas mikrovasküler endotel hücre izolasyon

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

Nörobilim

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.

Fare Direnç arterlerden mikrovasküler endotel Tüpler İzolasyonu

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.

Primer Fare Akciğer Endotel Hücrelerinin İzolasyonu

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

İmmünoloji ve Enfeksiyon

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 ...

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.

Murin Koroner Damar düz kas hücrelerinin izolasyonu

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.

Fare primer karaciğer sinüzoidal endotel hücrelerinin izolasyonu ve karakterizasyonu

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.

Tek Hücreli Multi-Omik Deneyler için Fare Karotis Arterlerinin Lümeninden Endotel Hücrelerinin İzolasyonu

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