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

Ci piace riconoscere Marcelo Toro per l&#39;assistenza con la citometria a flusso. Questo studio è stato sostenuto dalla svedese Heart-Lung Foundation e, Commissione UE FP7 &quot;CardioCell&quot;, Consiglio svedese della ricerca, AFA assicurazioni e ALF. OB è stato sostenuto da Deutsche Forschungsgemeinschaft.

1. Isolamento dei Nuclei Cardiac <ol><li> Coat tubi ultracentrifuga (Tubi centrifuga Beckman # 363664) con 10 ml di 1% BSA / PBS soluzione di rivestimento. Coprire le provette e farli ruotare per 30 minuti in un rotatore tubo. Rimuovere la soluzione di rivestimento e lasciare la centrifuga tubi aria secca (un tubo per il cuore di topo è richiesto per l&#39;analisi di cuore di topo singoli, alternativamente fino a 5 cuore di topo o 1 g di tessuto cardiaco da una specie diversa (ad esempio umano) può essere elaborato ...

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Identificazione accurata di nuclei cardiomiocita è cruciale per l&#39;analisi dei processi rigenerativi nel miocardio 2,3. Le tecniche convenzionali per isolare cardiomiociti di tessuto fresco si basa principalmente sulla digestione enzimatica delle proteine ​​della matrice extracellulare e la successiva purificazione dalle cellule interstiziali mediante centrifugazione a bassa velocità. Ulteriore purificazione di cardiomiociti viventi di cellule staminali embrionali (ESC) può essere eseguita da immuno...

<table border="1"><tbody><tr><td> 1. Lysis Buffer </td></tr><tr><td> Nome del reattivo </td></tr><tr><td> 0,32 M di saccarosio </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM CaCl 2 </td></tr><tr><td> 5 mM acetato di magnesio </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. Saccarosio tampone </td></tr><tr><td> Nome del reattivo </td></tr><tr><td> 2,1 M saccarosio </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM acetato di magnesio </td></tr><tr><td> 1 mM DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 3. Nuclei soluzione per la conservazione (NSB plus) </td></tr><tr&gt; <td> Nome del reattivo </td></tr><tr><td> 0,44 M di saccarosio </td></tr><tr><td> 10 mM Tris-HCl (pH = 7,2) </td></tr><tr><td> 70 mM KCl </td></tr><tr><td> 10 mM MgCl 2 </td></tr><tr><td> 1,5 mM spermina </td></tr></tbody></table><table border="1"><tbody><tr><td> Reagenti e materiale </td><td> Azienda </td></tr><tr><td> Isotipo IgG di coniglio ChIP-Grade, # ab37415 </td><td> Abcam </td></tr><tr><td> Coniglio anti-PCM-1 anticorpo, # HPA023374 </td><td> Atlas Anticorpi </td></tr><tr><td> Donkey sec. anticorpo anti-coniglio Alexa Fluor 488, # A-21.206 o equivalente sec. immunofluorescenza </td><td> Life Technologies </td></tr><tr><td> DRAQ5 </td><td> Biostatus </td></tr><tr><td> filtri cella 30 micron, 70 micron e 100 micron </td><td> BD Biosciences </td></tr><tr><td> Douncer vetro (40 ml) epestello &quot;L&quot; </td><td> VWR (Wheaton Industries Inc.) </td></tr><tr><td> T-25 Ultra-Turrax </td><td> IKA Germania </td></tr><tr><td> Disperdente strumento S25 N-18 G </td><td> IKA Germania </td></tr><tr><td> Centrifuga Beckman Avanti </td><td> Beckman Coulter </td></tr><tr><td> Provette Falcon da 15 ml e 50 ml </td><td> VWR </td></tr><tr><td> Tubi centrifuga Beckman # 363664 </td><td> Beckman Coulter </td></tr><tr><td> JS13.1 rotore oscillante libero </td><td> Beckman Coulter </td></tr><tr><td> Influx citometro </td><td> Beckman Coulter </td></tr><tr><td> Tubo Rotator </td><td> VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

Identificazione dei nuclei cardiomiociti è stato impegnativo in sezioni di tessuto, come la maggior parte delle strategie di contare solo su proteine ​​marker citoplasmatici 1. Eventi rari in miociti cardiaci quali la proliferazione e l&#39;apoptosi richiedono una identificazione accurata dei miociti cardiaci nuclei di analizzare il rinnovamento cellulare in omeostasi e in condizioni patologiche 2. Qui, forniamo un metodo per isolare nuclei cardiomyocyte dal tessuto post mortem di sedimentazione densità e immunomarcatura con anticorpi contro materiale pericentriolar 1 (PCM-1) e la successiva selezione citometria a flusso. Questa strategia permette una analisi un elevato throughput e l&#39;isolamento con il vantaggio di lavorare altrettanto bene su tessuti freschi e congelati materiale d&#39;archivio. Ciò rende possibile studiare materiale già raccolto in biobanche. Questa tecnica è applicabile e testato in una vasta gamma di specie e adatto a molteplici applicazioni a valle, come il carbonio-14 datazione 3, cell-cyCLE analisi 4, la visualizzazione di analoghi della timidina (ad esempio BrdU e IDU) 4, l&#39;analisi del trascrittoma ed epigenetici.

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

Nuclei cardiaca sono isolati tramite sedimentazione densità e immunolabeled con anticorpi contro materiale pericentriolar 1 (PCM-1) per identificare e ordinare nuclei cardiomiociti mediante citometria a flusso.

Isolamento dei Nuclei Cardiomyocyte dal post-mortem del tessuto

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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