验尸报告组织心肌细胞核分离
Summary
心肌细胞核通过密度沉淀分离与1 pericentriolar材料（PCM-1）识别和排序流式细胞仪检测心肌细胞核抗体immunolabeled。
Abstract
鉴定心肌细胞的细胞核已在组织切片挑战，因为大多数的战略仅依靠细胞质标记蛋白1。在罕见的事件，如心肌细胞增殖和凋亡需要的心肌细胞的细胞核的准确识别，分析细胞在病理条件下的稳态和重建。在这里，我们提供了一种方法来隔离验尸组织密度沉淀和免疫标记抗体对pericentriolar材料（PCM-1）和随后的流式细胞仪分选心肌细胞核。这一战略使一个高吞吐量的分析与新鲜组织和冰冻的档案材料，同样的工作的优势和隔离。这使人们有可能已经在生物银行收集研究材料。这项技术是适用和广泛的物种进行测试，适用于多种下游应用，如碳14约会3，细胞CYCLE分析，可视化胸腺嘧啶核苷类似物（如BrdU和IDU）4，转录组和表观遗传分析。
Protocol
1。分离心脏核外套超离心管（贝克曼离心管＃363664）10毫升1％BSA / PBS涂层解决方案。例管，并让他们旋转管旋转30分钟。删除的涂层解决方案，让空气干燥离心管（每管小鼠心脏单小鼠心脏，交替上升至5小鼠心脏或1克的心脏组织的分析，从不同的物种（如人类）可以处理在一管）。 以下所有步骤应在冰上进行。解剖用手术刀从新鲜或冷冻单元的小鼠心脏的左心室。请注意，此协?…
Discussion
心肌细胞核的准确鉴定为2,3在心肌再生过程的分析是至关重要的。主要是基于传统技术从新鲜组织分离的心肌细胞细胞外基质蛋白消化酶和随后的间质细胞，低速离心净化。从胚胎干细胞（ESC）的生活心肌的进一步净化可进行免疫标记表面标志物，如9或10 SIRPA线粒体染料，固定的心肌细胞可以识别如肌球蛋白重链（MHC）或核蛋白质，如细 胞质标记GATA4或Nkx2.5。然而，N…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢马塞洛·托罗用流式细胞仪的协助下。这项研究是由瑞典的心脏和肺脏基金会，欧盟委员会第七框架计划“CardioCell”，瑞典研究理事会，亚洲电影大奖保险和ALF的支持。转播是由德意志研究联合会的支持。
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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Cite This Article
Bergmann, O., Jovinge, S. Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue. J. Vis. Exp. (65), e4205, doi:10.3791/4205 (2012).