Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.
Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.
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DOI：
10.1038/nprot.2015.110
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发表时间：
2015-10
期刊：
影响因子：
14.8
通讯作者：
Rando TA
中科院分区：
文献类型：
--
作者：
Liu L;Cheung TH;Charville GW;Rando TA
纯化的干细胞群体的预期分离已经极大地改变了干细胞生物学领域，并且已经成为不同生物体中跨组织研究的主要焦点。肌肉干细胞现在是哺乳动物系统中研究最深入的干细胞群体之一，这些细胞的前瞻性分离允许细胞和分子表征，这在十年前是做梦也想不到的。在这个协议中，我们描述了如何分离肌肉干细胞从四肢肌肉的成年小鼠的荧光激活细胞分选（FACS）。我们提供了一个详细的描述，从肢体肌肉，一个程序，是必不可少的，以最大限度地提高细胞产量的单核细胞的物理和酶的解离。然后，我们描述了一种基于FACS的方法，用于获得静止或活化的肌肉干细胞（VCAM+/CD 31 −/CD 45 −/Sca 1 −）的纯群体。该方案还允许从肌肉组织中分离内皮细胞、造血细胞和间充质干细胞。
The prospective isolation of purified stem cell populations has dramatically altered the field of stem cell biology and has been a major focus of research across tissues in different organisms. Muscle stem cells are now among the most intensely studied stem cell populations in mammalian systems and the prospective isolation of these cells has allowed cellular and molecular characterizations not dreamed of a decade ago. In this protocol, we describe how to isolate muscle stem cells from limb muscles of adult mice by fluorescence-activated cell sorting (FACS). We provide a detailed description of the physical and enzymatic dissociation of mononucleated cells from limb muscles, a procedure that is essential to maximize cell yield. We then describe a FACS-based method for obtaining exquisitely pure populations of either quiescent or activated muscle stem cells (VCAM+/CD31−/CD45−/Sca1−). The protocol also allows for the isolation of endothelial cells, hematopoietic cells, and mesenchymal stem cells from muscle tissue.