Bölüm I. Hazırlık cerrahi için <ol><li> Rat cerrahi bir gün önce, tüm çözümler yapılır emin olun, ancak henüz herhangi bir enzimler katmayan. Tamponlar A, B ve yıkama tamponu depolama önce sterilite için filtre olmalıdır. Tamponlar 4 ° C&#39;de muhafaza edilmelidir </li><li> 6-iyi doku kültürü plakaları 10-20 mg / ml 2 ml 1XSFM laminin de önceki gün kaplanmış olmalıdır ve 37 ° C CO 2 kuvöz içinde saklanmalıdır. </li><li> Ameliyatın yapıldığı gün,% 70 etanol ile kelepçe, makas, forseps ve sterilizasyon cerrahi aletler hazırlamak ve onları kağıt havlu üzerinde kurumaya bırakın. </li><li> 0.5 mL Heparin Çözümü (90 U / ml) ile dolu bir şırınga 1 ml hazır hale getirilir. </li><li> Şimdi ters peristaltik pompa kullanarak,% 70 etanol ile çalışan perfüzyon aparatı yıkamak için iyi bir uygulamadır. Bir kez etanol ile durulama bitmiş cihazın, çift distile su ile temizleyin, durulayın ve akış sonunda, 37 ° C&#39;lik su banyosuna yerleştirilir bir beher toplanan Tampon A. ile durulayın. </li><li> Tampon A ve sol şırınga tüp 40 ml, 40 ml Tampon B. ile temizlenmelidir perfüzyon cihazı hazırlamak için doğru şırınga tüp doldurmak </li><li> Tampon bir aparat üzerinden akmasına izin vererek kateter ucu Başbakan perfüzyon tüp. Tampon, 40 ml seviyesine şırınga Dolum. Bu adımdan sonra tüp içinde sıkışıp hiçbir hava kabarcığı olmadığından emin olun. </li><li> Şimdi, perfüzyon boru Tampon B. ile astarlanmalıdır olduğunu tampon şırınga stopcock vanasını kapatın ve örneğin Tampon B işlemi tekrarlayın </li><li> Son olarak, perfüzyon aparat Tampon B valfli kapak açık kalan, ancak akışı ile IV hattı regülatör kullanarak durdu. </li><li> Toplama beher akış olsa tampon atın. </li><li> Ameliyat öncesi yapılacak en son şey, diğer çözümlerin bir gün önce filtre gibi enzim çözeltisi stok solüsyonu gerekli enzimleri eklemek ve daha sonra filtre. Süzülür sonra, bu çözüm oda sıcaklığında bırakılabilir. </li></ol> Bölüm II. Sıçan kalp diseksiyonu <ol><li> Takiben standart bir protokol, bir gaz odası İsofluranın ile sıçan euthanize. </li><li> % 70 etanol ile toraks kesi alanı sterilize edin. </li><li> Açık göğüs makas kullanarak ve kalp maruz. </li><li> 0.5 mL Heparin çözüm (90 U / ml) ile sol ventrikül enjekte edilir. </li><li> Buz Tampon B. aort sağlam, yerinde büyük bir kısmı kalp çıkarın </li></ol> Bölüm III. Miyositler izolasyon <ol><li> Aşağıdaki yordam, düzgün yapıştırılır ise tipik bir sıçan kalp çubuk şeklinde miyositler yüksek bir fraksiyonu (ölü yuvarlak hücrelere karşı) boyun eğmek zorundadır . </li><li> Başlamak için, kateter aort kelepçe. Kateterin ucu çok uzakta, koroner arter yoluyla kalbin iyi perfüzyon sağlamak için kalbin içine itti olmamalıdır. </li><li> Kelepçeli, bir dikiş ile kateter aort bağlar. </li><li> Sonra, hızlı bir damla oranı (~ 60 damla / dk) ve kalp yıkamak için izin Tampon B izin IV hattı regülatör açın. </li><li> Tampon B yıkama sırasında, kulakçıklar ve kalp yapışan herhangi bir yağ ya da akciğer dokusunda kaldırmak için küçük makas ve forseps kullanabilir. </li><li> Tampon B tamamlandıktan sonra, Tampon A. akım anahtarı </li><li> Tampon 5 dakika boyunca akmasına izin olsa da, birkaç küçük görevleri hızlı bir şekilde yapılmalıdır. </li><li> İlk olarak, 37 ° C su banyosunda Enzim Çözüm sıcak. </li><li> Son olarak, Tampon şırınga (ama hala mevcut boru) boşaldığında, şırınga Enzim Çözüm 50 ml yük bir perfüzyon aparat </li><li> Enzim Çözüm perfuses kalp kadar, artık tüm su banyosu (pipetleme) toplama beherine flow-through atmak için gerekli. </li><li> Enzim Çözüm kalp perfuses, şırınga A. doldurmak için toplama beher enzim çözeltisi aktarmak için kurulur peristaltik pompa aktive </li><li> Enzim çözeltisi, 10 dakika süreyle kalp akmasına izin verin. Kalp sindirir gibi, bu şişirilmiş aramaya başlar. </li><li> Şırınga içinde enzim çözeltisi 0,1 M CaCl 2 37.5 mcL ekleyin 0.1 mM Ca 2 etkili bir konsantrasyon elde etmek. </li><li> 10 dakika için 0.1 M CaCl 2 ek bir 50 mcL şırınga A ekleyerek 0.2 mM kalsiyum konsantrasyonunu artırmak ve perfüzyon bir 10 dakika daha devam edelim sonra. </li><li> Ventriküller kesilmiş ve 20 mL Enzim Çözüm içeren küçük bir steril beher transfer. </li><li> Beher 0,1 M CaCl 2 40 mcL daha ekleyerek 0.4 mM Kalsiyum konsantrasyonu artırır. </li><li> Yavaşça, küçük steril makas bir çift ile 10 veya daha fazla parçalar halinde kalp kıyma. </li><li> 37 5 dakika boyunca beher ° C&#39;de nazik sallanan. </li><li> 40 mL 0.1 M CaCl 20.6 mM Ca 2 + etkin konsantrasyon vermek ve kalp adet plastik bir transfer pipetlemeyin 3-5 kez yavaşça çiğnemek. </li><li> 37 ek bir 5 dakika inkübe sonra ° C, 40 ml 0,1 M CaCl 2 ve yavaşça eskisi gibi çiğnemek. </li><li> Ayrı sindirilir tek miyositler olmayan sindirilir steril 500 mikron mesh yoluyla filtrasyon kullanarak bağ dokusu. </li><li> Hücrelerin oda sıcaklığında 10 dakika, 50 ml tüp yerleşmek için izin verin. </li><li> Bir transfer pipetlemeyin ile süpernatant atın. </li><li> Yıkama Tamponu # 1 yavaşça hücreleri tekrar süspansiyon ve hücrelerin oda sıcaklığında 10-20 dk yerleşmek için izin. </li><li> Hücrelere yerleşen olsa da, bu kez ters bir mikroskop altında süspansiyon hücrelerinin kalite ve canlılığını değerlendirmek için bir fırsat olarak küçük bir kısım almak ve kullanmak. İyi bir hazırlık net desenli hücreleri gibi bir çubuk yüksek oranda (&gt;% 80) sahip olacak. </li><li> Hücreleri yerleştikten sonra, Yıkama Tamponu # 2 supernatant ve yavaşça tekrar süspansiyon hücreleri atın. Hücrelerin oda sıcaklığında dinlendiriniz, yine 10-20 dakika sürebilir ve süpernatantı atmak gerekir. </li><li> Yıkama işlemi tekrarlayın bir kez Yıkama Tamponu # 3, ve hücrelerin daha fazla kültür için hazır olacak. </li></ol> Bölüm IV. Miyosit hücre kültürü <ol><li> % 5 serum Medya yavaşça hücreleri tekrar süspansiyon haline getirin. Hücrelerin doku kültürü tabak aktarmadan önce, 1x SFM plakaları yıkayın. 3-5 saat boyunca CO 2 doku kültürü inkübatör istenen yoğunluk ve inkübe hücrelere aktarın. </li><li> 1x SFM medya geçin. </li><li> Hücreleri dört gün kadar kültürlü ve deneyler için gerekli olabilir. </li></ol> Bölüm V. Temsilcisi Sonuçlar / Sonuç Protokolü doğru yapılır inverted mikroskop altında% 70 den fazla canlı kalp miyositler olmamalıdır. <img src="/files/ftp_upload/1308/fig1.jpg" alt="Şekil 1" /> Tablo 1: 1 L Çözüm tarifi 10X Ca 2 +-KH Tampon: <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> kitle </td><td> Final Konsantrasyon </td></tr><tr><td> NaCl </td><td> 68,96 g </td><td> 1180 mM </td></tr><tr><td> KCl </td><td> 3.58 g </td><td> 48 mm </td></tr><tr><td> Hepes </td><td> 59.58 g </td><td> 250 mm </td></tr><tr><td> K 2 HPO 4 </td><td> 2.85 g </td><td> 12,5 mm </td></tr><tr><td> MgSO 4 </td><td> 3.08 g </td><td> 12,5 mm </td></tr></tbody></table> Tablo 2: 1 L Tampon A Çözüm tarifi: 100 ml 10X KH tampon 1.98 g glukoz ve glukoz ile yakın fizyolojisi durumu (~ 300) ozmolarite ayarlayın son hacmi 1 L. getirmek. NaOH ile pH 7.4 &#39;e getirin. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> - </td><td> Kitle </td><td> Final Konsantrasyon </td></tr><tr><td> 10X KH Tampon </td><td> 100 mL </td><td> 100 ml. </td></tr><tr><td> Glikoz </td><td> 1.98 g </td><td> 11 mm </td></tr><tr><td> 1 L Getir </td><td></td><td></td></tr><tr><td> NaOH ile pH 7.4 Getir </td><td></td><td></td></tr><tr><td colspan="3"> Glukoz ile yakın fizyolojik durumu (~ 300) ozmolarite ayarlayın. </td></tr></tbody></table> Tablo 3: Enzim Tamponlar Çözüm tarifi <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> kitle </td><td> Final Konsantrasyon </td></tr><tr><td> Kollajenaz Tip II </td><td> 25 mg </td><td> 0.05% (w / v) </td></tr><tr><td> Proteaz XIV. </td><td> 10 mg. </td><td> % 0,02 (w / v) </td></tr><tr><td> BDM </td><td> 0.025 g </td><td> 5 mM </td></tr><tr><td> Karnitin </td><td> 0.020 g </td><td> 2 mM </td></tr><tr><td> Boğa </td><td> 0.031 g </td><td> 5 mM </td></tr><tr><td> Glutamik asit </td><td> 0.020 g </td><td> 2 mM </td></tr><tr><td> 0.1 M CaCl 2 </td><td> 12.5 uL </td><td> 25 UM </td></tr><tr><td> Bir tampon </td><td></td><td> 50 ml doldurun </td></tr></tbody></table> Tablo 4: 25X BDM / Taurin / BSA (B / T / B) Çözüm tarifi <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td> kitle </td><td> Final Konsantrasyon </td></tr><tr><td> BDM </td><td> 0.076 g </td><td> 125 mm </td></tr><tr><td> Boğa </td><td> 0.094 g </td><td> 125 mm </td></tr><tr><td> BSA </td><td></td><td> % 0,1 (w / v) </td></tr><tr><td> Bir tampon </td><td></td><td> 6 ml doldurun </td></tr></tbody></table> Tablo 5: Tampon B ve Yıkama Tamponlar Çözüm tarifi <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Tampon: </td><td> B </td><td> # 1 </td><td> # 2 </td><td> # 3 </td></tr><tr><td> Ekle: </td><td></td><td></td><td></td><td></td></tr><tr><td> 0.5 M CaCl 2 </td><td> 0,4 mL </td><td> 0.1 mL </td><td> 0.125 ml </td><td> 0.15 mL </td></tr><tr><td> CaCl 2 Final konsantrasyonu </td><td> 1 mM </td><td> 1 mM </td><td> 1.25 mM </td><td> 1.5 mM </td></tr><tr><td> 25X B / T / B </td><td> --- </td><td> 2 mL </td><td> 2 mL </td><td> 2 mL </td></tr><tr><td> Nihai hacmi Tampon </td><td> 200 mL </td><td> 50 ml </td><td> 50 ml </td><td> 50 ml </td></tr></tbody></table> Tablo 6: Kültürleme Medya İçin Çözüm Tarif <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 2X Serum Medya (SFM) </td><td></td><td></td></tr><tr><td rowspan="2"> </td><td rowspan="2"></td><td> Final Konsantrasyon </td></tr><tr><td> (Seyreltme sonra) </td></tr><tr><td> Karnitin </td><td> 0.099 g </td><td> 10 mM (5 mM) </td></tr><tr><td> Boğa </td><td> 0.063 g </td><td> 10 mM (5 mM) </td></tr><tr><td> Kreatin </td><td> 0.075 g </td><td> 10 mM (5 mM) </td></tr><tr><td> Antimikotik / antibiyotik </td><td> 0.5 ml </td><td> % 1 (% 0.5) (v / v) </td></tr><tr><td> Medya 199 </td><td> 45 ml </td><td> 50 ml doldurun </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 1X SFM </td><td></td><td></td></tr><tr><td></td><td></td><td> Final Konsantrasyon </td></tr><tr><td> 2X SFM </td><td> 25 ml </td><td> 1X </td></tr><tr><td> Medya 199 </td><td></td><td> 50 ml doldurun </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 1X% 5 serum Medya </td><td></td><td></td></tr><tr><td></td><td></td><td> Final Konsantrasyon </td></tr><tr><td> 2X SFM </td><td> 25 ml </td><td> 1X </td></tr><tr><td> FBS </td><td> 2.5 mL </td><td> % 5 (v / v) </td></tr><tr><td> Medya 199 </td><td></td><td> 50 ml doldurun </td></tr></tbody></table>

<ol>
	<li>Miriyala, J., Nguyen, T., Yue, D. T., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CaVbeta+subunits,+and+lack+of+functional+reserve,+in+protein+kinase+A+modulation+of+cardiac+CaV1.2+channels.">Role of CaVbeta subunits, and lack of functional reserve, in protein kinase A modulation of cardiac CaV1.2 channels.</a> Circ Res. 102 (7), e54-e64 (2008).</li><li>SX, T. a. k. a. h. a. s. h. i., Mittman, S., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+modulatory+effects+of+five+human+auxiliary+beta2+subunit+splice+variants+on+L-type+calcium+channel+gating.">Distinctive modulatory effects of five human auxiliary beta2 subunit splice variants on L-type calcium channel gating.</a> Biophys. , 84-845 (2003).</li></ol>

Kritik bir adım, izole kalp perfüzyon sistemi üzerinde asılı olduğu hızı. Enzimatik sindirim sürenin uzunluğu, her bir sıçan için biraz farklı olabilir. Ayarı, kalp, sindirim düzenli bir süre sonra ne kadar yumuşak olur bağlıdır. Enzim sindirimi yavaş Ca 2 +-hoşgörülü sağlıklı hücreleri elde etmek için gerekli Ca 2 + sonra kurtarma. Hiyalüronidaz Proteaz XIV yerine kullanılır hariç kobay için, protokol ile aynıdır. Tipik olarak, onl...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S6191</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9541</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4034</td>
<td>&nbsp;</td>
<tr>
<td>K2HPO4</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9666</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>63068</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7021</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7902</td>
<td>&nbsp;</td>
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<td>Heparin Sodium Salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4784</td>
<td>&nbsp;</td>
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<td>Collagenase Type II</td>
<td>&nbsp;</td>
<td>Worthington Biochemical</td>
<td>LS004176</td>
<td>&nbsp;</td>
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<td>Protease XIV</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5147</td>
<td>&nbsp;</td>
<tr>
<td>2,3-Butanedione Monoxime (BDM)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B0753</td>
<td>&nbsp;</td>
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<td>Carnitine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C9500</td>
<td>&nbsp;</td>
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<td>Taurine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T0625</td>
<td>&nbsp;</td>
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<td>Glutamic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1501</td>
<td>&nbsp;</td>
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<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3294</td>
<td>&nbsp;</td>
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<td>Creatine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C3630</td>
<td>&nbsp;</td>
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<td>Antibiotic</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-009-CI</td>
<td>&nbsp;</td>
<tr>
<td>Media 199</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11150</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>354232</td>
<td>&nbsp;</td>
<tr>
<td>water bath</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-462-5</td>
<td>&nbsp;</td>
<tr>
<td>variable flow chemical pump</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-077-67</td>
<td>&nbsp;</td>
<tr>
<td>6-well culture plate</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3516</td>
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primary culture of adult rat heart myocytes

Kültüre birincil yetişkin kemirgen kalp hücrelerinin Kardiyovasküler araştırmalar için önemli bir model sistem. Bununla birlikte, sağlam, işlevsel kültür yetişkin miyositler kurulması birçok araştırmacı için, hız sınırlayıcı teknik açıdan zor bir adım olabilir. Burada, birkaç gün süreyle kültür canlı kalır yetişkin sıçan kalp miyositler yüksek verim elde etmek için bir protokol nitelendirdi. Kalp düşük Ca altında izole ve kollajenaz ve proteaz ile perfüze<sup&gt; 2 +</sup&gt; Koşulları tek miyositler kurtarmak için. Ca<sup&gt; 2 +</sup&gt; Dayanıklı hücrelerin ekstrasellüler Ca kademeli artışlar elde edilir<sup&gt; 2 +</sup&gt; Takip eden üç yıkama adımları konsantrasyon. Hücreler, süzülmüş, kültür ortamı yeniden süspanse ve laminin kaplı bordroları kaplama vardır. Bu protokol kullanılarak elde Kültüre miyositler dört gün için geçerli değildir ve elektrofizyoloji, biyokimya, görüntüleme ve moleküler biyoloji de dahil olmak üzere pek çok deneyler için uygundur.

Watch this Scientific Journal Video about Primary Culture of Adult Rat Heart Myocytes at JoVE.com

Primary Culture of Adult Rat Heart Myocytes

Bu çalışmada, yetişkin sıçan kalp miyositler izole ve kültürü tipik bir şekilde tanımladı. Kollajenaz ve proteaz tek miyositler sindirimi ve izole etmek için kullanılır. Miyositler Çoğu deney gereksinimlerini karşılamak bu protokolü takip kültürü.

Yetişkin Sıçan Kalp miyositler İlköğretim Kültür

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

primary-culture-of-adult-rat-heart-myocytes

Research

JoVE Journal

Biology

20.2K Görüntüleme.  Columbia University. Bu çalışmada, yetişkin sıçan kalp miyositler izole ve kültürü tipik bir şekilde tanımladı. Kollajenaz ve proteaz tek miyositler sindirimi ve izole etmek için kullanılır. Miyositler Çoğu deney gereksinimlerini karşılamak bu protokolü takip kültürü.

Video: Primary Culture of Adult Rat Heart Myocytes

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

Yetişkin Tavşan Retina Organotipik Kültürü

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

Biyoloji

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Sıçan Kalp Engineered Doku implantasyonu

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

Konfokal Görüntüleme İzolasyon ve Yetişkin kardiyak miyositler Genetik Manipülasyon

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

Kemirgenler itibaren İlköğretim Yetişkin Fibroblast Kültürleri kurulması

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

Mitokondri Yüksek Coupled Sıçan Kalp hazırlanması

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Sıçan Karaciğer Hücreleri İzolasyon ve İlköğretim Kültür

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Yetişkin fareler ile Sinoatriyal Düğüm miyositlerin İzolasyonu, Kültür ve Fonksiyonel Karakterizasyonu Yöntemleri

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

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 and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Yenidoğan ve Erişkin Fare Ciltlerindeki Primer Fare Keratinositlerin İzolasyonu ve Kültürü

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

Sıçan Adrenokortikal hücreleri ve deneyleri Steroidogenic fonksiyonların birincil kültür

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...