Lim Chai Ling, Ling King-Hwa, Cheah Pike-See
Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
J Biol Methods. 2018 Mar 16;5(1):e87. doi: 10.14440/jbm.2018.219. eCollection 2018.
Satellite cells are myogenic cells responsible for muscle growth shortly after birth and muscle repair/regeneration during adulthood. Therapies based on satellite cells hold promise for treating muscular dysfunctions. Studying satellite cells is technically challenging owing to their low abundance, small size and anatomical dispersed location between the basal lamina and the sarcolemma of myofibers. In this article, we present three improved protocol strategies for studying the properties of satellite cells of the mouse during the different stages of muscle regeneration: (1) immunostaining of freshly isolated single myofibers to facilitate the study of quiescent satellite cells, (2) cultivation of single myofibers on Matrigel-coated dish to study the myogenesis programs initiated by satellite cell activation, and (3) cultivation of single myofibers in floating conditions to analyze activated satellite cells or the doubling time of satellite cells in myofibers. In brief, when compared to previously published protocols, this article presented an improved protocol that requires shorter experimental time and less laborious approach for higher yield of intact single myofibers for downstream analyses.
卫星细胞是生肌细胞，在出生后不久负责肌肉生长，并在成年期负责肌肉修复/再生。基于卫星细胞的疗法有望治疗肌肉功能障碍。由于卫星细胞丰度低、体积小且在肌纤维基膜和肌膜之间的解剖位置分散，研究卫星细胞在技术上具有挑战性。在本文中，我们提出了三种改进的方案策略，用于研究小鼠在肌肉再生不同阶段卫星细胞的特性：（1）对新鲜分离的单个肌纤维进行免疫染色，以促进对静止卫星细胞的研究；（2）在基质胶包被的培养皿上培养单个肌纤维，以研究卫星细胞激活引发的肌生成程序；（3）在悬浮条件下培养单个肌纤维，以分析激活的卫星细胞或肌纤维中卫星细胞的倍增时间。简而言之，与之前发表的方案相比，本文提出了一种改进的方案，该方案所需实验时间更短，操作更简便，能获得更高产量的完整单个肌纤维用于下游分析。
