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

Vi vil gerne anerkende, Marcelo Toro for hjælp med flowcytometri. Denne undersøgelse blev støttet af den svenske Heart-og Lunge Foundation, EU-Kommissionen FP7 &quot;CardioCell&quot;, svensk Forskningsråd, AFA forsikringer og ALF. OB blev støttet af Deutsche Forschungsgemeinschaft.

1. Isolering af Cardiac Kerner <ol><li> Coat ultracentrifugerør (Beckman centrifugerør # 363664) med 10 ml 1% BSA / PBS coatingopløsning. Hætten rørene og lad dem rotere i 30 minutter i et rør rotator. Fjerne coatingopløsning og lade centrifugeglas lufttørre (et rør pr mus hjertet kræves til analyse af enkelte mus hjerter, skiftevis op til 5 mus hjerte eller 1 g af hjertevæv fra en anden art (fx menneske) kan behandles i et rør). </li><li> Alle følgende trin bør udføres på is. Dissekere den venstre ven...

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Nøjagtig identifikation af cardiomyocyte kerner er afgørende for analysen af regenerative processer i myokardiet 2,3. Konventionelle teknikker til isolering cardiomyocytter fra frisk væv er primært baseret på enzymatisk nedbrydning af ekstracellulære matrixproteiner, og efterfølgende oprensning af interstitielle celler ved centrifugering ved lav hastighed. Yderligere oprensning af levende cardiomyocytter fra embryoniske stamceller (ESC) kan udføres ved immunolabeling med overflademarkører såsom Sirp...

<table border="1"><tbody><tr><td> 1. Lysis Buffer </td></tr><tr><td> Navn på reagenset </td></tr><tr><td> 0,32 M sucrose </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM CaCl2 </td></tr><tr><td> 5 mM magnesiumacetat </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. Sucrosepuffer </td></tr><tr><td> Navn på reagenset </td></tr><tr><td> 2,1 M saccharose </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM magnesiumacetat </td></tr><tr><td> 1 mM DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 3. Kerner opbevaringspuffer (NSB plus) </td></tr><tr&gt; <td> Navn på reagenset </td></tr><tr><td> 0,44 M sucrose </td></tr><tr><td> 10 mM Tris-HCI (pH = 7,2) </td></tr><tr><td> 70 mM KCI </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> Reagenser og udstyr </td><td> Firma </td></tr><tr><td> Isotype kanin IgG-CHIP Grade, # ab37415 </td><td> Abcam </td></tr><tr><td> Kanin-anti-PCM-1-antistof, # HPA023374 </td><td> Atlas Antistoffer </td></tr><tr><td> Æsel sek. antistof, anti-kanin Alexa 488 Fluor, # A-21.206 eller tilsvarende sek. fluorescerende antistof </td><td> Life Technologies </td></tr><tr><td> DRAQ5 </td><td> Biostatus </td></tr><tr><td> celle sier 30 um, 70 um og 100 um </td><td> BD Biosciences </td></tr><tr><td> Glas douncer (40 ml) ogpistil &quot;L&quot; </td><td> VWR (Wheaton Industries Inc.) </td></tr><tr><td> T-25 Ultra-Turrax homogenisator </td><td> IKA Tyskland </td></tr><tr><td> Dispergerings værktøj S25-N-18 G </td><td> IKA Tyskland </td></tr><tr><td> Beckman Avanti Centrifuge </td><td> Beckman Coulter </td></tr><tr><td> Falcon-rør 15 ml og 50 ml </td><td> VWR </td></tr><tr><td> Beckman-centrifuge rør # 363664 </td><td> Beckman Coulter </td></tr><tr><td> JS13.1 frit svingende rotor </td><td> Beckman Coulter </td></tr><tr><td> Tilgangen cytometeret </td><td> Beckman Coulter </td></tr><tr><td> Tube Rotator </td><td> VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

Identifikation af cardiomyocyte kerner har været udfordrende i vævssnit, som de fleste strategier er baseret udelukkende på cytoplasmiske markørproteiner 1. Sjældne begivenheder i hjertemyocytter såsom spredning og apoptose kræver en præcis identifikation af hjertets myocytter kerner til at analysere cellulære fornyelse i homøostase og i patologiske tilstande 2. Her, tilvejebringer vi en fremgangsmåde til isolering cardiomyocyte kerner fra post mortem væv ved tæthed sedimentation og immunolabeling med antistoffer mod pericentriolar materiale 1 (PCM-1) og efterfølgende flowcytometri sortering. Denne strategi muliggør et højt gennemløb analyse og isolering med fordelen ved at arbejde lige så godt på frisk væv og frosset arkivmateriale. Dette gør det muligt at studere materialet allerede er indsamlet i biobanker. Denne teknik kan anvendes og testes i en lang række arter og er egnet til flere efterfølgende anvendelser, såsom carbon-14 datering 3, celle-cyCle analyse 4, visualisering af thymidinanaloger (f.eks BrdU og IDU) 4, transkriptom og epigenetiske analyser.

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

Cardiac kerner isoleres via densitet sedimentation og immunolabeled med antistoffer mod pericentriolar materiale 1 (PCM-1) til at identificere og sortere cardiomyocyte kerner ved flowcytometri.

Isolering af cardiomyocyte Kerner fra Post-mortem Tissue

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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