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

Nous tenons à remercier Marcelo Toro pour l&#39;aide à la cytométrie en flux. Cette étude a été soutenue par le Suédois coeur et du poumon Fondation, la Commission européenne FP7 &quot;CardioCell&quot;, Conseil de recherche suédois, AFA assurances et ALF. OB a été soutenu par la Deutsche Forschungsgemeinschaft.

1. Isolement des noyaux cardiaque <ol><li> Tubes ultracentrifugeuse Coat (Tubes à centrifuger Beckman # 363664) avec 10 ml de solution de revêtement de 1% de BSA / PBS. Reboucher les tubes et les laisser tourner pendant 30 min dans un rotateur tube. Retirer la solution de revêtement et de laisser la centrifugeuse tubes d&#39;air sec (un tube par cœur de la souris est nécessaire pour l&#39;analyse des cœurs de souris, en alternance jusqu&#39;à 5 cœurs de souris ou 1 g de tissu cardiaque d&#39;une espèce diffé...

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L&#39;identification précise des noyaux des cardiomyocytes est crucial pour l&#39;analyse des processus de régénération dans le myocarde 2,3. Les techniques conventionnelles pour isoler les cardiomyocytes des tissus frais sont principalement basées sur la digestion enzymatique des protéines de la matrice extracellulaire et la purification ultérieure à partir de cellules interstitielles par centrifugation à faible vitesse. Une purification supplémentaire des cardiomyocytes vivant à partir de cellule...

<table border="1"><tbody><tr><td> 1. Tampon de lyse </td></tr><tr><td> Nom du réactif </td></tr><tr><td> 0,32 M de saccharose </td></tr><tr><td> 10 mM de Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM de CaCl 2 </td></tr><tr><td> 5 mM d&#39;acétate de magnésium </td></tr><tr><td> 2,0 mM d&#39;EDTA </td></tr><tr><td> 0,5 mM EGTA </td></tr><tr><td> DTT 1 mM </td></tr></tbody></table><table border="1"><tbody><tr><td> 2. Tampon saccharose </td></tr><tr><td> Nom du réactif </td></tr><tr><td> 2,1 M de saccharose </td></tr><tr><td> 10 mM de Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM d&#39;acétate de magnésium </td></tr><tr><td> DTT 1 mM </td></tr></tbody></table><table border="1"><tbody><tr><td> 3. Tampon de stockage des noyaux (NSB plus) </td></tr><tr&gt; <td> Nom du réactif </td></tr><tr><td> 0,44 M de saccharose </td></tr><tr><td> 10 mM de Tris-HCl (pH = 7,2) </td></tr><tr><td> 70 mM de KCl </td></tr><tr><td> 10 mM MgCl 2 </td></tr><tr><td> 1,5 mM de spermine </td></tr></tbody></table><table border="1"><tbody><tr><td> Réactifs et appareillage </td><td> Entreprise </td></tr><tr><td> Isotype IgG de lapin-ChIP Grade, # ab37415 </td><td> Abcam </td></tr><tr><td> De lapin anti-PCM-1 anticorps, # HPA023374 </td><td> Anticorps Atlas </td></tr><tr><td> Ane sec. un anticorps anti-lapin Alexa 488 Fluor, # A-21206 ou l&#39;équivalent sec. immunofluorescence </td><td> Life Technologies </td></tr><tr><td> DRAQ5 </td><td> Biostatus </td></tr><tr><td> crépines de cellules de 30 um, 70 um et 100 um </td><td> BD Biosciences </td></tr><tr><td> Verre douncer (40 ml) etpilon &quot;L&quot; </td><td> VWR (Wheaton Industries Inc) </td></tr><tr><td> T-25 Ultra-Turrax Homogénéisateur </td><td> IKA Allemagne </td></tr><tr><td> Outil de dispersion S25 N-18 G </td><td> IKA Allemagne </td></tr><tr><td> Beckman Avanti Centrifugeuse </td><td> Beckman Coulter </td></tr><tr><td> Falcon tubes 15 ml et 50 ml </td><td> VWR </td></tr><tr><td> Tubes à centrifuger Beckman # 363664 </td><td> Beckman Coulter </td></tr><tr><td> JS13.1 rotor oscillation libre </td><td> Beckman Coulter </td></tr><tr><td> Afflux cytomètre </td><td> Beckman Coulter </td></tr><tr><td> Rotator Tube </td><td> VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

Identification des noyaux des cardiomyocytes a été difficile dans des coupes de tissus comme la plupart des stratégies de compter uniquement sur ​​les protéines marqueurs cytoplasmiques 1. Les événements rares dans les myocytes cardiaques tels que la prolifération et l&#39;apoptose nécessite une identification précise des noyaux des myocytes cardiaques pour analyser le renouvellement cellulaire dans l&#39;homéostasie et dans des conditions pathologiques 2. Ici, nous fournissons une méthode pour isoler les noyaux de cardiomyocytes post-mortem des tissus par la sédimentation de densité et immunomarquage avec des anticorps contre le matériel péricentriolaire 1 (PCM-1) et après le tri par cytométrie en flux. Cette stratégie permet une analyse à haut débit et l&#39;isolement avec l&#39;avantage de travailler aussi bien sur les tissus frais et congelés documents d&#39;archives. Cela permet d&#39;étudier des documents déjà recueillis dans des biobanques. Cette technique est applicable et testé dans un large éventail d&#39;espèces et adapté à de multiples applications en aval tels que le carbone-14 datant 3, cellule-cyarticle analyse 4, la visualisation des analogues de la thymidine (BrdU par exemple et les UDI) 4, l&#39;analyse du transcriptome et épigénétiques.

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

Noyaux cardiaque sont isolés par sédimentation de densité et immunomarquées avec des anticorps contre matériel péricentriolaire 1 (PCM-1) d&#39;identifier et de trier les noyaux des cardiomyocytes par cytométrie de flux.

Isolement des noyaux de cardiomyocytes post-mortem des tissus

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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