Name: isolation of cardiomyocyte nuclei from post-mortem tissue
Uploaded: 2012-07-10T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

Biz sitometrisi ile yardım için Marcelo Toro kabul etmek istiyorum. Bu çalışma, İsveç ve Kalp-Akciğer Vakfı, AB Komisyonu 7.ÇP &quot;CardioCell&quot;, İsveç Araştırma Konseyi, AFA sigortalar ve ALF tarafından desteklenmiştir. OB Deutsche Forschungsgemeinschaft tarafından desteklenmiştir.

1. Kardiyak Çekirdek İzolasyonu <ol><li> % 1 BSA / PBS kaplama solüsyonu 10 ml Coat ultrasantrifüjdeki tüpler (Beckman santrifüj tüpünde # 363.664). Tüpler Cap ve onları bir tüp rotator 30 dakika döndürün. Kaplama çözümü çıkarın ve santrifüj tüplerine hava kuru (fare kalp başına bir tüp farklı canlı türlerinin tek bir fare kalpleri, dönüşümlü olarak 5&#39;e kadar fare kalpleri veya kalp dokusunun 1 gr analizi için gerekli izin (örneğin insan) işlenebilir bir tüp içinde). </li><l...

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Kardiyomiyosit çekirdeklerinin doğru tespit miyokard 2,3 rejeneratif süreçlerinin analizi için çok önemlidir. Taze dokudan kardiyomiyositler izole etmek için, geleneksel teknikler özellikle hücre dışı matris proteinlerine enzimatik sindirimi ve düşük hızlı santrifüjleme ile interstisyel hücreleri daha sonra saflaştırma dayanmaktadır. Embriyonik kök hücreler (ESC) ile yaşayan kardiyomiyositlerin saflaştırma gibi Sirpa 9 veya mitokondriyal boyalar 10 gibi yüzey...

<table border="1"><tbody><tr><td> 1. Lizis Tampon </td></tr><tr><td> Reaktif Adı </td></tr><tr><td> 0,32 M sakaroz </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM CaCl2 </td></tr><tr><td> 5 mM magnezyum asetat </td></tr><tr><td> 2.0 mM EDTA </td></tr><tr><td> 0.5 mM EGTA </td></tr><tr><td> 1 mM DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 2. Sakkaroz tampon </td></tr><tr><td> Reaktif Adı </td></tr><tr><td> 2.1 M sakkaroz </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM magnezyum asetat </td></tr><tr><td> 1 mM DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 3. Çekirdekler depolama tamponu (NSB plus) </td></tr><tr&gt; <td> Reaktif Adı </td></tr><tr><td> 0,44 M sakaroz </td></tr><tr><td> 10 mM Tris-HCl (pH = 7,2) </td></tr><tr><td> 70 mM KCl </td></tr><tr><td> 10 mM MgCl2 </td></tr><tr><td> 1,5 mM spermin </td></tr></tbody></table><table border="1"><tbody><tr><td> Reaktifler ve Ekipmanları </td><td> Şirket </td></tr><tr><td> Izotip tavşan IgG-ChIP Grade # ab37415 </td><td> Abcam </td></tr><tr><td> Tavşan anti-PCM-1 antikoru, # HPA023374 </td><td> Atlas Antikorlar </td></tr><tr><td> Eşek sn. antikoru, anti-tavşan Alexa 488 Fluor, # A-21206 veya eşdeğer san. floresan antikor </td><td> Yaşam Teknolojileri </td></tr><tr><td> DRAQ5 </td><td> Biostatus </td></tr><tr><td> Hücre süzgeçler 30 mikron, 70 mikron ve 100 mikron </td><td> BD Biosciences </td></tr><tr><td> Cam douncer (40 ml) ve&quot;L&quot; havaneli </td><td> VWR (Wheaton Sanayi A.Ş.) </td></tr><tr><td> T-25 Ultra-Turrax Homojenizatör </td><td> IKA Almanya </td></tr><tr><td> Aracı S25 N-18 G Dispergatör </td><td> IKA Almanya </td></tr><tr><td> Beckman Avanti Santrifüj </td><td> Beckman Coulter </td></tr><tr><td> Falcon tüpler 15 ml ve 50 ml </td><td> VWR </td></tr><tr><td> Beckman Santrifüj Tüpleri # 363664 </td><td> Beckman Coulter </td></tr><tr><td> JS13.1 ücretsiz sallanan rotor </td><td> Beckman Coulter </td></tr><tr><td> Akını sitometresi </td><td> Beckman Coulter </td></tr><tr><td> Tüp Rotator </td><td> VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

En stratejileri sadece sitoplazmik protein işaretleyicileri 1 güveniyor gibi kardiyomiyosit çekirdeklerinin belirlenmesi doku kesitlerinde zor olmuştur. Gibi çoğalması ve apoptosis gibi kardiyak miyositler Nadir olaylar kardiyak miyosit çekirdeklerinin doğru bir tanımlama homeostazında ve patolojik durumların 2 hücresel yenilenme analiz gerektirir. Burada, pericentriolar malzeme 1 (PCM-1) ve sonraki akım sitometri sıralama karşı antikorlar ile yoğunluğu sedimantasyon ve immün tarafından otopsi doku kardiyomiyosit çekirdekleri izole etmek için bir yöntem sağlar. Bu strateji, taze doku ve dondurulmuş arşiv malzemesi gayet iyi çalışma avantajı ile yüksek verimlilik analizi ve izolasyon sağlar. Bu da zaten biyobankalar toplanan malzeme çalışma kolaylaştırır. Bu teknik uygulanabilir ve türler, geniş bir aralık içinde test edilmiştir ve karbon-14 3, hücre-CY olarak birden çok alt-uygulamalar için uygundurcle analizi 4, timidin analogları görselleştirme (örneğin BrdU ve DUK) 4, transcriptome ve epigenetik analizi.

Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Kardiyak çekirdeklerin yoğunluğu sedimentasyon ile izole edilmiş ve akım sitometri kardiyomiyosit çekirdeklerinin belirlemek ve sıralamak için pericentriolar malzeme 1 (PCM-1) karşı antikorlar ile immunolabeled edilir.

Post-mortem Doku gelen kardiyomiyosit Çekirdeklerin İzolasyonu

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

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