Hücre ve Gelişim Biyolojisi çekmesi ekipman Bölümü kullanımı Marc Wozniak. Vanderbilt Üniversitesi Hücre Görüntüleme Paylaşımlı Kaynak. Bu çalışma DMB için ERP AHA hibe 12PRE10950005, DMB için AHA hibe 11GRNT7690040, NIH hibe R01 HL037675 tarafından desteklenmektedir. DMB Kardiyovasküler Araştırma Gladys P. Stahlman Başkanıdır.

Hayvanları kapsayan tüm işlemler uygun hayvan kullanımı ile onaylanmasını sağlayın ve vücut bakımı. 1. Advance Stok Tamponlar hazırlayın <ol><li> 2 L&#39;stok Ca2 + içermeyen Tyrode (Tablo 1) hazırlayın. NaOH ile 7.4 &#39;e pH ayarlayın. </li><li> 2 L&#39;stok ve 1.2 mM Ca2 + Tyrode (Tablo 2) hazırlayın. NaOH ile 7.4 &#39;e pH ayarlayın. </li></ol> Deney gününde 2. Perfüsvonun, Transferi ve Sindirim Tamponlar hazırlayın <ol><li> Ca 2 + Tyrode (Tablo 3), 150 ml perfüzyon tamponu hazırlayın ve bir 0.2 mikron filtre içinden süzme. </li><li> 35 ml hazırlayın Ca 2 + bedelsiz &quot;Tampon A&quot; &quot;Tampon B&quot; (Tablo 5) içeren (Tablo 4) ve 25 ml Ca 2 +. 0.2 mikron filtre aracılığıyla her filtre. </li><li> 0 çözümler hazırlayın.06, 0.24, 0.6, ve karıştırma Tampon A 1.2 mM Ca 2 + ve B Tablo 6 başına. </li><li> Fare vücut ağırlığı (BW) göre 25 mi perfüzyon tamponu (Tablo 7) olarak sindirim enzimleri eritilerek 25 mi sindirim tamponu hazırlayın. Kullanılmadan önce bir 0.2 mikron filtre içinden süzme. </li></ol> 3. Deney Düzeneği <ol><li> Mevcut piyasada bulunan ya da 3 ml&#39;lik bir akış oranı izin vermek için bir peristaltik pompa, boru ve bir ısı değiştirici sargı kullanılarak, sabit bir akış Langendorff cihazına monte / 37 ° C&#39;ye kadar ısıtıldı perfüzyon dak. Basit şematik Bell tarafından 2011 gözden sağlanan, ark 18 edilir. </li><li> Kanül dışarı akış 3 mL / dakikalık bir akış hızında 37 ° C üstünde olacak şekilde, Langendorff aparatındaki dolaşan su banyosu sıcaklığı (~ 47 ° C) ayarlayın. </li><li> Ultra saf tip I, 100 ml su, ardından 15 dakika boyunca perfüzyon sistemine,% 70 etanol ile çalıştırın. Sistemli Run perfüzyon tampon5 dakika boyunca m hava kabarcıklarını ortadan kaldırılmaktadır. </li><li> İstediğiniz bir yöntem ile tüm cerrahi aletleri sterilize. </li><li> Bir 23 G Luer adaptörü saplama ucu PE-50 boru kısa bir parça (yaklaşık 3 mm) takılarak kanül oluşturun. Boru ısı ucu aort yerleştirilmiş olacak, üzerinde bir dudak oluşturmak için. </li></ol> 4. kardiyomiyosit İzolasyon <ol><li> Intraperitoneal enjeksiyon ile, 0.2 ml heparin solüsyonu (1000 lU / ml) ile fare enjekte edilir. </li><li> 5 dakika sonra, izofluoran fare uyutmak. Tam (güçlü ayak tutam hiçbir yanıt) anestezi yaparken,% 70 etanol ile göğüs püskürtün. , Göğüs açın çabuk 4 ° C perfüzyon tamponunda aorta ve yer zarar vermemek için dikkatli olmak, kalbini kesip. Kavramak ve kalp altında kaldırmak için kavisli bir forseps kullanın. Bu ince makas ile kalbin arkasında ekleri kırpmaya alan oluşturur. </li><li> Yavaşça ince forseps iki çift aort tutup ve dikkatli bir şekilde openi çekerek kalp Cannulatekanülün dudak aort ng. Bu koroner damar yoluyla kalbin perfüzyon inhibe gibi, kanül ucu aort kapağı geçmiş ve ventrikül içine olmadığından emin olun. </li><li> Hemen kanülün dudak üzerinde aorta çevresinde 5-0 ipek sütür bir döngü bağlayarak kanüle aorta edin. </li><li> Not: 4.4 ile 4.2 mümkün olduğunca çabuk en kaliteli sindirim için tamamlanmalıdır Adımlar. </li><li> Not: Kalp daha önce perfüzyon tampon akan Langendorff cihazına takılı bir kanül üzerine direkt olarak monte edilebilir. Bununla birlikte, bir teşhir mikroskopu altında perfüzyon tamponu küçük bir şınngaya bağlanmış bir kanül üzerine kalp asılı çok başarılı olduğu bulunmuştur. Bu perfüzyon tamponu bir miktar koroner arterlerin temizlenmesi için takip ve güvenli bir şekilde kanül bağlı kalp sağlanması vasıtasıyla itilmesine izin verir. Sonra kanül perfüzyon tampon çalışan Langendorff&#39;a asılır. </li><li> Perfuse2-3 dakika boyunca 3 ml / dakikalık bir akış hızında serbest Ca2 + perfüzyon tamponu ile kalp. </li><li> Sindirim tampon enzim ve 7-10 dakika boyunca serpmek geçmek, kalp renkli yumuşak ve hafif olacak. </li><li> Not: Sindirim zaman enzim aktivitesi ve kalp büyüklüğüne göre ayarlanabilir. </li><li> Kalp palpably sarkık sonra, Tampon A (Ca 2 + bedelsiz tamponu) ile steril bir p60 çanak kanül ve yerden kalp kaldırmak. </li><li> Atriumlarm ve büyük damarları çıkarın. İsterseniz sağ ventrikül çıkarın. Forseps ile, küçük parçalar halinde ventrikül ayrı. 5 ml steril bir transfer pipet ile birkaç kez pipet başka hücreleri dağıtmak için. </li><li> 250 um naylon ağ filtre içerisinden 50 ml konik tüp içine hücre süspansiyonu filtre edin. </li><li> Süspansiyon, yaklaşık 10 dakika boyunca pelet izin verin. </li><li> 0.06 mM Ca 2 + çözeltisi içine pelet aktarın. Hücreler, 10 dakika boyunca pelet izin verin. En ölü hücreler yüzer olurken Sağlıklı hücreler, pelet olacaktır. </li&gt; <li> Süpernatanı havalandırın, ve 0.24 mM Ca 2 + çözeltisine pelet aktarın. </li><li> 10 dakika inkübasyon, aspirasyon tekrarlayın ve hücreler 1.2 mM çözelti haline gelene kadar kalan iki Ca 2 + çözümleri ile aktarın. Bu noktada, hücrelerin çoğunluğunun çubuk şekilli miyositler olmalıdır. </li></ol> 5. Ölçme Kasılma ve Kalsiyum Geçişler <ol><li> Not: kontraklil ve kalsiyum geçici Hem eş zamanlı olarak ölçülebilir. </li><li> 1 mg / ml arasında bir konsantrasyonda susuz DMSO içinde yeniden süspanse edilerek Fura-02:00 çözelti hazırlayın. Fura-02:00 solüsyonu, -20 ° C&#39;de bir desikatör içinde saklanabilir. </li><li> Yük 1 ul Fura-02:00 ile suspensiyon halindeki hücreler 1 ml. Hücreler koyu 10 dakika sonra aspire yüzer oturmak için izin verin. Hücreler yıkandı ve yıkamalar arasında 10 dakika boyunca karanlıkta pelet sağlayan, Ca 2 + Tyrode çözeltisi ile iki kez yıkayın. </li><li> Isıtmalı pe sağlayan bir sahne prospektüste bir cam lamelBir miyositlerdeki alan uyarıcı ile volta rfusion / aspirasyon ve elektrot. Ters mikroskop yerleştirin sahne insert bir immersiyon yağı hedefi kullanıyorsanız lens petrol bir damla ekleyerek, floresan ölçümü için ayarlanmış. </li><li> 1.2 mM Ca 2 + tirod çözeltisi ısıtmalı yerçekimi perfüzyon kurmak sahnede çözelti 37 ° C ulaşır böylece. Vakum aspirasyon takın. Elektrotlar takın. </li><li> Mikroskop sistemi açın ve rasyometrik floresan ve hücre boyutlandırma veri toplama yazılımı açın. </li><li> An perfüzyon engelleme sağlıklı hücreleri yerleşmek için izin, sahne inserte Fura-02:00 yüklü hücrelerin birkaç damla ekleyin. </li><li> Istenen hücreyi bulmak için 40X objektif kullanın. Sağlıklı bir hücre çubuk şeklinde ve kendiliğinden sözleşme olmamalıdır. Bu gözle görülür ve eşit sözleşme cep 5-20 V&#39;de tempolu gerektiği zaman </li><li> Not: Fizyolojik analizi, yüksek kaliteli parçalanıp sindirilmesi gerekmektedir. Bu sağlıklı görünüyorsun ama pa yok hücrelere sahip olmaları mümkündür<html> fizyolojik analizi için de ce. Sindirim protokol, yüksek kaliteli, sağlıklı miyositleri almak için arıtma gerekebilir. Ayrıca, mutant veya hastalıklı kalpleri protokol değişiklikleri gerektirebilir. </li><li> Objektif rotasyon ve değişim diyafram böylece istenilen hücre ekran ve arka plan üzerinde yatay dizilmiş olan Ayarı görünür değil. </li><li> Miyositinin her sonuna kadar kenar algılama barlar hizalayın ve eşiği ayarlayın. Düzgün sarkomer ile hücrenin bir kısmı üzerinde sarkomer algılama hizalayın. Artık size bu bar yapabilirsiniz, sarkomer uzunluğunun daha doğru matematiksel model bu kadar uzun sarkomer iki popülasyonları üzerinde değil gibi olacaktır. </li><li> Pace hücreleri 2 Hz ve kayıt önce 10-15 saniye boyunca 5-20 V. </li><li> Kayıt izleme. Ortam ışığı iyi kalsiyum geçici kayıt için minimize edilmelidir. </li></ol> 6. Analiz <ol><li> Rasyometrik floresan ve hücre boyutlandırma veri toplama yazılımı kullanarak izlerini analiz edin. </li></ol>title &quot;&gt; 7. Diğer Çalışmalar <ol><li> Fiksasyon ve immün, doğrudan ilaç etkileri, kısa vadeli kültürü ve atomik kuvvet mikroskobu değerlendirirken: Bu protokol dahil olmak üzere diğer deneyler çeşitli için kullanılabilir fazla hücreleri sağlar. </li></ol>

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Biz ifşa hiçbir şey yok.

Kalite kardiyomiyositlerin izolasyonu uygulama ve optimizasyon bazı düzeyi gerektirir. Bu protokol büyük ölçüde sindirim sonucunu etkileyecek önemli adımları içermektedir. Performans ve protokol giderirken bu dikkatle düşünülmelidir. Kanülasyon ve perfüzyon için kalp kaldırılması süre az, ideal olarak, beş dakikadan daha aza düşürülmesi gerekmektedir. Buz soğukluğunda tamponda kalp fare çıkarılması ve kesme ve kanüle sonra buz soğukluğunda perfüzyon tampon maddesi içinde hemen kalp durulama da kaliteli bir sindirim şansını arttıracaktır. Kanülasyon kendisi kritik bir adımdır. Bu koroner arterlerin perfüzyonu önleyecek şekilde kanül ucu değil, ventrikül içine aort kapağı içinden aortun ilk dalları altına yerleştirilmelidir. Koroner perfüzyon kaliteli bir sindirim için gereklidir. Bu laboratuvarda, kanül perfüzyon tamponu ile doldurulmuş bir şırınga takılır. Yavaşça bir küçük enjekteMontaj ve kan net koroner arterler kanül yeterliliğini test edebilirsiniz izledikten sonra perfüzyon tamponu l miktarı. Bu protokolü Sorun Giderme dikkatle yukarıda belirtilen kritik adımları değerlendirilmesini içermektedir. Kalp, sindirim sırasında hissedilir yumuşak hale gelmezse, birkaç potansiyel nedenleri vardır. İlk olarak, kanülasyon sırasında kanülün ucunun, koroner arterlerin ile perfüzyon önlenmesi, aort kapağı çok ötesine olabilir. Bu Langendorff cihazı üzerine kalp asılı ancak bir şırınga ile, kanül yoluyla, perfüzyon tamponuna bir miktar itme kan ve berrak, koroner arterlerin dikkatle takip önce değerlendirilebilir. Perfüzyon yeterli ama eğer kalp, iyi sindirmek enzimlerin farklı partileri satın dikkate almaz. Enzimler aslında karışımlar olduğundan, bir sürü aynı hareket yok. Her enzimin küçük miktarlarda satın öneririz. Çok, purc iyi sindirmek olduğu tespit edildiğindeileride kullanmak için bu çok büyük miktarlarda hase. Tartışıldığı gibi kalp diseksiyonu ve kanulasyonun daha uzun 5-10 dakika sürer, eğer sindirilmiş hücrelerin kalitesi kötü olabilir. Nakavt veya tedavi edilen hayvanlarla geçmeden önce diseksiyonu master yabani türü veya tedavi edilmemiş hayvanlar ile tamamlayan izolasyonların düşünün. Sindirimden sonra, hücreler kalsiyum yavaşça dayanıklı yapılmalıdır gerekir. Hücreler, kalsiyum çözeltinin her birinde en az 10 dakika harcamak gerekir. Bu adımları acele hücrelerin verimini ve yaşayabilirliğini de azaltabilir. Tekrarlanabilir de, fizyolojik ölçümler kaliteli bir sindirme ile başlayan bağlıdır. Ölçmek için hücreleri seçerken, kendiliğinden bir sızdıran, hasarlı hücre zarı gösterir, sözleşme değildir hücreleri seçin. Tempolu zaman Hücreler eşit sözleşme olmalıdır. Kasılma tutarlılığı sağlamak için ölçümler kayıt önce 10-15 sn için hücreleri kalp pili düşünün. Hücre görünürse eşit daralan, fakat CO &#39;ölçümlerintraction büyüklüğü büyük ölçüde zamanla, ilk tanımlanan ilgi bölgesi sarkomer sadece bir nüfusu kapsayan emin olmak için kontrol edin değişir. Bazen iki hücre birbirine grubu ve faiz (ROI) bölgelerinden birisi olarak sarkomerinde iki farklı popülasyonlarını ölçmeye olabilir. Uygun bir ROI seçilmiş fakat büzülme tek bir hücre içinde değişken ise, hücre boyama olabilir ve bir başka hücre seçilmelidir. Kalsiyum geçici için, su işlevsel mevcuttur Fura-02:00 miktarını değiştirir gibi kurutulur susuz DMSO Fura-02:00 sulandırmak için kritik öneme sahiptir. Yükleme işleminden sonra, bütün Fura-02:00 hücre içinde de-esterleştirmek izin vermek için birinci yıkama ve ölçüm geçici arasında en az 20 dakika izin verir. Herhangi bir floresan boya gibi, sırasında ve yüklemeden sonra ışıktan hücreleri korumak. Bazı hücreler, böylece arada bir kalsiyum geçici ölçülü bir hücrede gözlenecektir boya, alıma olmayacaktır. Kontraklil ve kalsiyum geçici kopyalamaları &quot;görünsedüzenleme hızı, büyük ölçüde (0.5-1 Hz) azalır temiz &quot;ölçümler yakın fare kalp atış hızı, tercih edilen en azından 2 Hz, böylece bir hızda bir hızda daha fazla fizyolojik bulunmaktadır. Arzu edildiği takdirde yalıtım kendisi değiştirilebilir. Bu laboratuar perfüzyon sırasında sabit bir akış yöntemi (fareler için 3 ml / dakika) kullanır. Bazı gruplar bir yerçekimi daha etkili akış bulduk. Sindirim daha kolay kalp gibi akış hızı artar sindirilir olarak görselleştirilebilen için yerçekimi akış perfüzyon sağlar. Bahsedildiği gibi, bu teknikler sınırlamaları taşırlar yapmak. Fizyolojik ölçümler kalitesi sindirim kalitesine bağlı olduğundan, her deney koşulu için birden fazla kalp miyositleri ölçmek için akıllıca olur. Her kalp ölçmek için birçok miyositleri sağlayacaktır, fakat tekrar sindirimler sonuçları kalpler arasında tutarlı olmasını sağlayacaktır. Deney tasarımı foll içeriyorsa İzolasyon, böylece, bir terminal işlemdirzamanla bir hayvanın kalp fonksiyonu nedeniyle yalnızca bir kez kardiyomiyositlerde hasat edilebilir. Daha farenin yüzden gerekli bireysel farenin kardiyomiyositlerin analiz kurban ederken bir kohort uzunlamasına takip edilebilir. Bizim odak bir nakavt fare kontraktiliteyi ve hücre iskeleti yapısı okuyor olmuştur, ancak, izole kardiyomiyositler farmakolojik etkilerini inceleyen, basınç veya hacim aşırı farelerden kardiyomiyositlerin okuyan, t-tübülleri için canlı hücre boyama gibi diğer deneyler çeşitli için kullanılabilir Tedavi, immunofloresan, transfeksiyon 9 ve transkripsiyonel 19 profilleme. Bu çok yönlü bir tekniktir kalp yetmezliği incelemek için kullanılan diğer yöntemlere göre, sayısız avantajlar sağlar. Tek tek hücrelerin incelenmesi fizyolojisi tüm organ f eşlik hücresel değişiklikler içgörü sağlayarak, bu tür ekokardiyografi veya elektrokardiyografi gibi tüm organ çalışmalar tarafından sunulan değildir bilgi verirailure. Bütün hücre kolayca boyanmış ve görüntüsü olabilir Buna ek olarak, izole edilmiş hücreler, görüntüleme alt-hücresel yapılar için daha üstündür. Kontraktilite, kalsiyum geçici ve immünofloresans analizi için kardiyomiyositlerin Ayırma deneysel tasarımlar çok çeşitli değerli bilgiler sunan çok yönlü bir tekniktir.

Cardiomyocytes provide the contractile force for the heart. Each myocyte contains organized cytoskeletal and contractile elements essential to contraction. The heart not only contains cardiomyocytes but also fibroblasts, connective tissue, and modified myocytes such as Purkinje fibers. Changes in any or all of these cell types can be seen in heart failure, making determination of the ultimate cause of heart dysfunction difficult. Decreased ventricular contractility of a failing heart is preceded by varying degrees of fibrous tissue build-up and hypertrophy and dysfunction of the cardiomyocytes1. Arrhythmias can be caused by dysfunction in the conducting cells2, fibrosis interrupting cardiac conduction3, or changes in the expression of ion channels in cardiomyocytes1,4. Indeed, these problems often co-exist in a complicated fashion.
Isolating and studying cardiomyocytes allows one to examine contractile and electrical dysfunction of individual myocytes in the context of overall heart function. Techniques to isolate individual cardiac myocytes were developed over 40 years ago in an effort to better study physiology of this &#8216;workhorse&#8217; cell of the heart. Isolation techniques have been improved to increase yield and quality of cells, with the vital development of coronary perfusion via cannulation of the aorta, first performed in 19705. This article describes cardiomyocyte isolation followed by measurement of contractility and calcium transients using ratiometric fluorescence and cell dimensioning data acquisition software. In both genetic and environmental models of heart disease, utilizing this protocol provides key information about cell contractility, sarcomeric contractility and relaxation, calcium transients, and cytoskeletal disruption. This system is often used to test the effect of drugs on isolated cardiomyocytes6,7. We employ the system to compare contractility and calcium transients of cardiomyocytes between wild type mice and mice with a mutation leading to dilated cardiomyopathy.
Studying individual cardiac myocytes has numerous advantages over other commonly used methods to study cardiac anatomy and physiology. Echocardiography and electrocardiography provide information about the contractile function and electrical conduction of the heart overall. Neither of these methods explains the cause or nature of the dysfunction below the level of the whole organ. Measuring contractility of individual cardiomyocytes using edge-detection and sarcomere length algorithms can demonstrate that changes within the contractile cell of the heart are present within global heart dysfunction. Measuring calcium transients demonstrates the influence of changes in ion flux at a cellular level to contractile dysfunction or arrhythmias.
In addition to complementing whole organ studies, this protocol provides cells easily stained for sub-cellular components. It is possible to see cytoskeletal changes by staining tissue sections of the whole heart; however, one is only able to visualize a cross section of cells. A whole cardiomyocyte is thicker than the typical tissue section, and given the arrangement and length of the cardiomyocytes, it is difficult to see whole cells in a single section. Isolated cardiomyocytes can be fixed immediately and stained for a variety of cytoskeletal elements and ion channels. Confocal images can be compiled into full thickness z-stacks of the cell. Additionally, staining isolated cardiac myocytes allows for staining of components that are impossible to visualize in sections, such as the membrane invaginations known as t-tubules.
Cultured cell lines of cardiac myocytes do exist, some of which have similar transcriptional profiles and phenotypic features to cardiac myocytes. Some, such as the HL-1 cell line, even retain some sarcomeric organization and rudimentary contractile ability8. Despite these qualities, cultured cells lack the &#8220;box car&#8221; shape and t-tubule structure of cardiomyocytes and have less sarcomeric organization. These components are essential to the function of cardiomyocytes in vivo. In addition, using cultured cells allows genetic manipulation but not in vivo physiologic manipulation. Isolated cardiomyocytes retain their cellular organization long enough to study but are still easily imaged and even transduced9. 
Isolated cardiomyocytes are extremely versatile, providing a substrate to identically analyze cells from varied experimental conditions. Isolation and physiological analysis of myocytes have been used by countless labs for a variety of experiments including studying the effect of drugs6,7 or small molecules10, environmental stressors11, infection12, illness13, or genetic mutation14,15 on contractility and/or calcium transients and studying the regulation of contractility and calcium release within a cardiomyocyte15-17. With the application of any of these stressors, changes in heart function could be due to any combination of changes in the cardiomyocytes themselves and changes in the surrounding environment of the heart, such as scarring, changes in conductivity, or changes to the extracellular matrix. This is an ideal protocol for answering research questions regarding individual myocyte structure and function in the context of heart failure from any cause.
Although it has countless advantages over other techniques, physiological analysis of isolated cardiomyocytes is not ideal for every research question. Isolation is a terminal procedure, thus cardiomyocytes can only be assessed at one time in the mouse&#8217;s life. This can be partially overcome using cohorts and sacrificing individual mice along the course of illness development or by monitoring the mouse to determine whether it has clinically significant heart failure before isolation. Although this protocol can determine whether cardiomyocyte dysfunction has occurred, one cannot draw the conclusion that this dysfunction is the root cause of heart failure without additional experimental information. It is possible that cardiomyocyte dysfunction itself is secondary to other changes in the heart. Despite these challenges, studying cardiomyocytes offers valuable information in determining the nature of dysfunction in the heart, especially when combined with other experiments. 
The following protocol is adapted from one provided by Dr. Chee Lim, Vanderbilt University. Although contractility and calcium flux data are highly reproducible and useful in comparing hearts with genetic or environmental manipulation, isolation itself remains a technique dependent protocol, requiring optimization. Minimizing the time between removing the heart from the mouse and perfusing the heart via aortic cannulation is essential for a quality digestion. In addition, digestion time and enzyme concentration can be optimized for the highest quality myocytes. Steps likely requiring optimization, along with suggestions for optimization, are noted below.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">NaCl</td>
			<td style="width:221px;">RPI</td>
			<td style="width:159px;">S23020</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">KCl</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">P-3911</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:253px;">MgCl2</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">M-8266</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">HEPES</td>
			<td style="width:221px;">EM Science</td>
			<td style="width:159px;">S320</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:253px;">NaH2PO4</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">S5011</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:253px;">CaCl2 &#376; 2H2O</td>
			<td style="width:221px;">Fisher</td>
			<td style="width:159px;">C79</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">D-(+)-Glucose</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">G7528</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">2,3-Butanedione-monoxime (BDM)</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">B-0753</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Taurine</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">T-0625</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">A-7030</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Collagenase B</td>
			<td style="width:221px;">Roche Diagnostics</td>
			<td style="width:159px;">1-088-823</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Collagenase D</td>
			<td style="width:221px;">Roche Diagnostics</td>
			<td style="width:159px;">1-088-882</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Protease XIV</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">P-5147</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Heparin</td>
			<td style="width:221px;">APP Pharmaceuticals, LLC</td>
			<td style="width:159px;">401586D</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Isofluorane</td>
			<td style="width:221px;">Butler Schein</td>
			<td style="width:159px;">11695-6776-2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">Fura-2 AM</td>
			<td style="width:221px;">Teflabs</td>
			<td style="width:159px;">103</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">DMSO &#8211; anhydrous</td>
			<td style="width:221px;">Sigma</td>
			<td style="width:159px;">276855</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:253px;">IonOptix cell pacing and fluorescent analysis system</td>
			<td style="width:221px;">IonOptix</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">250 um nylon mesh filter</td>
			<td style="width:221px;">Sefar America</td>
			<td style="width:159px;">Lab Pak 03-250/50</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">23G leur-stub adaptor</td>
			<td style="width:221px;">Becton Dickinson</td>
			<td style="width:159px;">1482619E</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:253px;">PE-50 tubing</td>
			<td style="width:221px;">Becton Dickinson</td>
			<td style="width:159px;">427410</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and physiological analysis of mouse cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Kontraklil ve geçici kopyalamaları (Şekil 2) toplandığında, verileri kolayca uygun yazılım ile analiz edilir. Bütün izleme, veya bunların kısımları (Şekil 3) ortalama edilebilir. Kasılma çeşitli yollarla analiz edilebilir. Sistolik fonksiyonu için, bir kısalma zirve fraksiyonel kısalma, ya da zamanla daralma hızı ile kasılma büyüklüğünü ve daralma hızı değerlendirmek. Diyastolik fonksiyon% 50 kısalma ve gevşeme hızı zaman ile benzer şekilde analiz edilebilir. Kalsiyum geçişlerini, benzer veri taban ve tepe Fura-2 oranları ve zaman zirve ya da başlangıca (Tablo 8) dahil olmak üzere mukayese edilebilir. Bu analizler benzer tüm organ disfonksiyonu olan bir kalp değişmiş olabilir herhangi biri sistolik ve diyastolik hücresel kasılma fonksiyonu WT ve KO kalpler, yanı sıra kalsiyum akı, karşılaştırılabilir veriler sağlamaktadır. Ayrıca, aynı izolasyonu hücresi olabilirimmunohistokimyasal lekeleme (Şekil 4) da dahil olmak üzere, protokolde sıralanan diğer deneyler için de kullanılabilir. <table border="0" cellpadding="0" cellspacing="0" width="352"><tbody><tr><td> Ca2 + içermeyen Tyrode </td><td> mM </td><td> FW / yoğunlaştırma </td><td> Miktar, 2 L çözeltisi için </td></tr><tr><td> NaCl </td><td> 135 </td><td> 58.44 g </td><td> 15.8 g </td></tr><tr><td> KCl </td><td> 4 </td><td> 74.56 g </td><td> 0.596 g </td></tr><tr><td> MgCI2 </td><td> 1 </td><td> 1 M </td><td> 2 mi </td></tr><tr><td> Hepes </td><td> 10 </td><td> 238.31 g </td><td> 4.77 g </td></tr><tr><td> NaH&#39;den 2 PO 4 </td><td> 0.33 </td><td> 141.96 g </td><td> 0.094 gr </td></tr></tbody></table> Tablo 1. Kalsiyum içermeyen Tyrode çözeltisi - ReageHisse senedi Ca 2 + -ücretsiz Tyrode çözeltisi 2 L NTS. <table border="0" cellpadding="0" cellspacing="0" width="352"><tbody><tr><td> 1.2 mM Ca 2 + Tirod </td><td> mM </td><td> FW / yoğunlaştırma </td><td> Miktar, 2 L çözeltisi için </td></tr><tr><td> NaCl </td><td> 137 </td><td> 58.44 g </td><td> 16 g </td></tr><tr><td> KCl </td><td> 5.4 </td><td> 74.56 g </td><td> 0.805 gr </td></tr><tr><td> MgCI2 </td><td> 0.5 </td><td> 1 M </td><td> 1 mi </td></tr><tr><td> Hepes </td><td> 10 </td><td> 238.31 g </td><td> 4.77 g </td></tr><tr><td> CaCl2 · 2H 2 O </td><td> 1.2 </td><td> 147.01 g </td><td> 0.353 gr </td></tr></tbody></table> Tablo 2. 1.2 mM Kalsiyum Tirode çözümü - stokunun 1.2 mM Ca 2 L Reaktifler 2+Çözelti. <table border="0" cellpadding="0" cellspacing="0" width="505"><tbody><tr><td> Perfüzyon Tampon </td><td> mM </td><td> FW </td><td> 150 ml Ca miktarı 2 + ücretsiz Tirod </td></tr><tr><td> Glikoz </td><td> 10 </td><td> 180.16 g </td><td> 0.27 gr </td></tr><tr><td> 2,3-Butandion-monoxime (BDM) </td><td> 10 </td><td> 101.11 g </td><td> 0.152 g, </td></tr><tr><td> Taurin </td><td> 5 </td><td> 125.16 g </td><td> 0.094 gr </td></tr></tbody></table> Tablo 3. Perfüzyon Tamponu - Reaktifler deney gününde perfüzyon tamponu hazırlamak için. <table border="0" cellpadding="0" cellspacing="0" width="388"><tbody><tr><td> Tampon A </td><td> Perfüzyon tamponu 35 mi </td></tr><tr><td> Sığır serum albümini (BSA) </td><td> 0.175 g </td></tr></tbody></table> Tablo 4. Tampon A - Reaktifler deney gününde Tampon A hazırlamak. <table border="0" cellpadding="0" cellspacing="0" width="448"><tbody><tr><td> Tampon B </td><td> mM </td><td> FW </td><td> Ca 2 + 25 mi Tyrode tamponu </td></tr><tr><td> Glikoz </td><td> 5 </td><td> 180.16 g </td><td> 0.0225 g </td></tr></tbody></table> Tablo 5. Tampon B - Reaktifler deney gününde Tampon B hazırlamak. <table border="0" cellpadding="0" cellspacing="0" width="299"><tbody><tr><td> Buffer Transferi </td><td> Tampon A (mi) </td><td> Tampon B (mi) </td></tr><tr><td> 0.06 mM Ca 2 + </td><td> 9.5 </td><td> 0.5</td></tr><tr><td> 0,24 mM Ca 2 + </td><td> 8 </td><td> 2 </td></tr><tr><td> 0.6 mM Ca 2 + </td><td> 5 </td><td> 5 </td></tr><tr><td> 1.2 mM Ca 2 + </td><td> 0 </td><td> 10 </td></tr></tbody></table> Tablo 6. Aktarım Tamponu - Reaktifler deney gününde transfer tamponu hazırlamak. <table border="0" cellpadding="0" cellspacing="0" width="341"><tbody><tr><td> Enzim Sindirim Tampon </td><td> Perfüzyon tamponu 25 mi </td></tr><tr><td> Kolajenaz B </td><td> 0.4 mg / g vücut ağırlığı </td></tr><tr><td> Kolajenaz D </td><td> , 0.3 mg / g vücut ağırlığı </td></tr><tr><td> Proteaz XIV </td><td> 0,05 mg / g vücut ağırlığı </td></tr></tbody></table> Tablo 7. Enzim Sindirim Tampon - Reaktifler enzim di hazırlamakDeney gününde gestion tamponu. <table border="0" cellpadding="0" cellspacing="0" width="246"><tbody><tr><td> Kasılma </td><td></td></tr><tr><td> Baseline </td><td> 1.73 mikron </td></tr><tr><td> Tepe </td><td> 1.64 mikron </td></tr><tr><td> Fraksiyonel kısaltılması </td><td> 4.70% </td></tr><tr><td> Zaman zirve </td><td> 0.056 sn </td></tr><tr><td> % 50 başlangıca Zaman </td><td> 0.043 sn </td></tr><tr><td> Kalsiyum geçici </td><td></td></tr><tr><td> Baseline </td><td> 1.18 </td></tr><tr><td> Tepe </td><td> 1.32 </td></tr><tr><td> Zaman zirve </td><td> 0.021 sn </td></tr><tr><td> % 50 başlangıca Zaman </td><td> 0.083 sn </td></tr></tbody></table> Tablo 8. Kasılma ve kalsiyum geçici analiz eder. K = 5. Deneysel tasarım 1. Genel bakış Şekil. A) Kalp enzim sindirim tamponu. B) koroner perfüzyon yoluyla sindirilir Hücreleri bireysel, sağlıklı miyositlere dağıtılır. Hücreleri kalsiyum hoşgörülü. Ge böylece haline C) Hücreler sırasıyla artan kalsiyum konsantrasyonları ile aktarılır) Hücreler bağımlı kalsiyum ile yüklenir boya, Fura-2 AM düzenlenmiştir. D) Yüklü hücreleri elektrik sarkomer uzunluğu, hücre boyu, ve floresan kaydedilir ise tempolu. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51109/51109fig1highres.jpg" target="_blank">, bu rakamın büyük bir versiyonunu görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 2" src="/files/ftp_upload/51109/51109fig2highres.jpg" /> Şekil 2. A) Örnek contracinfertilitedir vs zamanı. vs zaman Fura-2 oranı tarafından temsil B) Örnek kalsiyum geçici sarkomer uzunluğu ile temsil trasesin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51109/51109fig2highres.jpg" target="_blank">, bu rakamın büyük bir versiyonunu görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 3," src="/files/ftp_upload/51109/51109fig3highres.jpg" /> Şekil 3. A) analizi. B&#39;den sonra Ortalama kontraktilitesi izleme) analizinden sonra kalsiyum geçici ortalaması. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51109/51109fig3highres.jpg" target="_blank">, bu rakamın büyük bir versiyonunu görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 4" src="/files/ftp_upload/51109/51109fig4highres.jpg" /> Kardiyomyosit sitoiskeleti (α-aktinin) Şekil 4. Immunofluorescent boyama.

Watch this Scientific Journal Video about Isolation and Physiological Analysis of Mouse Cardiomyocytes at JoVE.com

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Fare kardiyomiyositlerde İzolasyon ve Fizyolojik Analizi

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

isolation-and-physiological-analysis-of-mouse-cardiomyocytes

Nanyang Technological University

Research

JoVE Journal

Biology

22.9K Görüntüleme.  Vanderbilt University.  Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause. 

Video: Isolation and Physiological Analysis of Mouse Cardiomyocytes

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

Yetişkin Mouse Hipokampal dentat girus diseksiyonu

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Biyoloji

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo Görüntüleme ve Yumurtalık Kanseri Ortotopik Fare Modeli Tedavi Tedaviler

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Yetişkin Fare kardiyomiyositlerde Tek Hücre transkripsiyonel Profilleme

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

Mürin Atriyal ve Ventriküler kardiyomiyositlerde izolasyonu ve Kv Kanal Kayıtları

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

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 and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

İzolasyon ve Kültür Yetişkin Fare Hücre Sinyal için kardiyomiyositlerin ve<em&gt; In vitro</em&gt; Kalp hipertrofisi

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

İzolasyon ve Kültür Hücrelerinin ve Fare Doku gelen Mitokondri Fonksiyonel Analizi

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

İzolasyon, Kültür ve Yetişkin Fare kardiyomiyositlerde İletimi

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

Bir Yetişkin Sıçan Kalpten Yüksek Kalitede Kardiyomiyosit, Endotel Hücresi ve Fibroblastların Eşzamanlı İzolasyonu

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably identify nuclei of cardiomyocytes on histological sections, many groups rely on isolating viable cardiomyocytes prior to fixation to perform immunostaining. However, these live cardiomyocyte isolation techniques require optimization to maximize the yield, viability and quality of the samples, with inherent fluctuations from sample to sample despite maximum optimization. Here, we report a reproducible protocol, involving fixation prior to enzymatic digestion of the heart, which leads to maximum yield while preserving the in vivo morphology of individual cardiomyocytes. We further developed an automated analysis platform to determine the number of nuclei and DNA content per nucleus for individual cardiomyocytes. After exposing the chest cavity, the heart was arrested in diastole by perfusion with 60 mM KCl in PBS. Next, the heart was fixed in 4% paraformaldehyde (PFA) solution, and then digested with 60 mg/mL collagenase solution. After digestions, cells were singularized by trituration, and the cardiomyocyte fraction was enriched via differential centrifugation. Isolated cardiomyocytes were stained for Troponin T and &#945;-actinin to assess purity of the obtained population. Furthermore, we developed an image analysis platform to determine cardiomyocyte nucleation and ploidy status following DAPI staining. Image based ploidy assessments led to consistent and reproducible results. Thus, with this protocol, it is possible to preserve native morphology of individual cardiomyocytes to allow immunocytochemistry and DNA content analysis while achieving maximum yield.

The goal of this work is to develop a method to reproducibly isolate cardiomyocytes from the adult heart and measure DNA content and nucleation.

İmmünsitokimya ve Ploidi Analizi için Sabit Kalplerden Kardiyomiyositlerin İzolasyon

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably ...