Isolering af cardiomyocyte Kerner fra Post-mortem Tissue
Summary
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.
Abstract
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.
Protocol
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi vil gerne anerkende, Marcelo Toro for hjælp med flowcytometri. Denne undersøgelse blev støttet af den svenske Heart-og Lunge Foundation, EU-Kommissionen FP7 "CardioCell", svensk Forskningsråd, AFA forsikringer og ALF. OB blev støttet af Deutsche Forschungsgemeinschaft.
Materials
| 1. Lysis Buffer |
| Name of the reagent |
| 0.32 M sucrose |
| 10 mM Tris-HCl (pH = 8) |
| 5 mM CaCl2 |
| 5 mM magnesium acetate |
| 2.0 mM EDTA |
| 0.5 mM EGTA |
| 1 mM DTT |
| 2. Sucrose buffer |
| Name of the reagent |
| 2.1 M sucrose |
| 10 mM Tris-HCl (pH = 8) |
| 5 mM magnesium acetate |
| 1 mM DTT |
| 3. Nuclei storage buffer (NSB plus) |
| Name of the reagent |
| 0.44 M sucrose |
| 10 mM Tris-HCl (pH = 7.2) |
| 70 mM KCl |
| 10 mM MgCl2 |
| 1.5 mM spermine |
| Reagents and Equipment | Company |
| Isotype rabbit IgG- ChIP Grade, #ab37415 | Abcam |
| Rabbit anti-PCM-1 antibody, #HPA023374 | Atlas Antibodies |
| Donkey sec. antibody, anti-rabbit Alexa 488 Fluor, #A-21206 or equivalent sec. fluorescent antibody | Life Technologies |
| DRAQ5 | Biostatus |
| cell strainers 30 μm, 70 μm and 100 μm | BD Biosciences |
| Glass douncer (40 ml) and pestle “L” | VWR (Wheaton Industries Inc.) |
| T-25 Ultra-Turrax Homogenizer | IKA Germany |
| Dispersing tool S25 N-18 G | IKA Germany |
| Beckman Avanti Centrifuge | Beckman Coulter |
| Falcon Tubes 15 ml and 50 ml | VWR |
| Beckman Centrifuge Tubes #363664 | Beckman Coulter |
| JS13.1 free swinging rotor | Beckman Coulter |
| Influx cytometer | Beckman Coulter |
| Tube Rotator | VWR |
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