Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells.
Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells.
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DOI：
10.1007/978-1-62703-128-8_28
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Yablonka-Reuveni, Zipora
中科院分区：
文献类型：
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作者：
Keire, Paul;Shearer, Andrew;Shefer, Gabi;Yablonka-Reuveni, Zipora
多核肌纤维是骨骼肌的功能性收缩单位。在成人肌肉中，位于肌纤维的基膜和质膜之间的单核卫星细胞是主要的肌源性干细胞。本章描述了从小鼠骨骼肌中分离、培养和免疫染色肌纤维的方法。肌纤维被完整分离并保留其相关的卫星细胞。第一个协议讨论了从屈趾短肌（FDB）肌肉的肌纤维分离。使用血清替代培养基，在涂有PureCol胶原蛋白（以前称为Vitrogen）的培养皿中培养这些短肌纤维。采用这样的培养条件，卫星细胞保持与肌纤维相关联，在肌纤维表面上经历增殖和分化。第二个协议讨论了较长的肌纤维的隔离趾长伸肌（EDL）肌肉。与其中多个肌纤维一起加工的FDB制备不同，较长的EDL肌纤维通常使用富含生长因子的培养基在涂覆有基质胶的皿中单独加工和培养。在这些条件下，卫星细胞最初保持与亲本肌纤维结合，随后迁移离开，产生增殖和分化的后代。也可以使用本文所述的方案分离和分析来自其他类型的肌肉（例如横膈膜、咬肌和眼外肌）的肌纤维。总的来说，分离的肌纤维培养物为研究亲本肌纤维及其相关卫星细胞之间的相互作用提供了必要的工具。本章提供了背景，程序和试剂更新，以及FDB和EDL肌肉分离的分步图像，未包括在我们2005年的本系列出版物中。
Multinucleated myofibers are the functional contractile units of skeletal muscle. In adult muscle, mononuclear satellite cells, located between the basal lamina and the plasmalemma of the myofiber, are the primary myogenic stem cells. This chapter describes protocols for isolation, culturing and immunostaining of myofibers from mouse skeletal muscle. Myofibers are isolated intact and retain their associated satellite cells. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are cultured in dishes coated with PureCol collagen (formerly known as Vitrogen) using a serum replacement medium. Employing such culture conditions, satellite cells remain associated with the myofibers, undergoing proliferation and differentiation on the myofiber surface. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. Different from the FDB preparation, where multiple myofibers are processed together, the longer EDL myofibers are typically processed and cultured individually in dishes coated with Matrigel using a growth factor rich medium. Under these conditions, satellite cells initially remain associated with the parent myofiber and later migrate away, giving rise to proliferating and differentiating progeny. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular muscles can also be isolated and analyzed using protocols described herein. Overall, cultures of isolated myofibers provide essential tools for studying the interplay between the parent myofiber and its associated satellite cells. The current chapter provides background, procedural, and reagent updates, and step-by-step images of FDB and EDL muscle isolations, not included in our 2005 publication in this series.