Chen Shujie, Ding Hongrong, Yao Xiangping, Xie Liwei
State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangdong Institute of Microbiology, Guangdong Academy of Sciences, Guangdong 510070, China.
Zhujiang Hospital, Southern Medical University, Guangdong 510282, China.
Bio Protoc. 2019 Jul 20;9(14):e3313. doi: 10.21769/BioProtoc.3313.
Myofiber isolation followed with culture could recapitulate and visualize satellite cells (SCs) activation, proliferation, and differentiation. This approach could be taken to understand the physiology of satellite cells and the molecular mechanism of regulatory factors, in terms of the involvement of intrinsic factors over SCs quiescence, activation, proliferation and differentiation. Single myofiber culture has several advantages that the traditional approach such as FASC and cryosection could not compete with. For example, myofiber isolation and culture could be used to observe SCs activation, proliferation and differentiation at a continuous manner within their physiological "niche" environment while FACS or cryosection could only capture single time-point upon external stimulation to activate satellite cells by BaCl, Cardiotoxin or ischemia. Furthermore, transfection with siRNA or overexpression vector could be performed under culture to understand the detailed molecular function of a specific gene on SCs physiology. With these advantages, the physiological state of SCs could be analyzed at multiple designated time-points by immunofluorescence staining. In this protocol, we provide an efficient and practical protocol to isolate single myofiber from EDL muscle, followed with culture and immunostaining.
肌纤维分离并进行培养能够概括并可视化卫星细胞（SCs）的激活、增殖和分化过程。就内在因素对卫星细胞静止、激活、增殖和分化的影响而言，这种方法可用于了解卫星细胞的生理学特性以及调节因子的分子机制。单根肌纤维培养具有一些传统方法（如荧光激活细胞分选术（FASC）和冰冻切片）所无法比拟的优势。例如，肌纤维分离和培养可用于在其生理“微环境”中连续观察卫星细胞的激活、增殖和分化，而FACS或冰冻切片只能在通过氯化钡、心肌毒素或缺血等外部刺激激活卫星细胞后捕获单个时间点的情况。此外，在培养过程中可以进行小干扰RNA（siRNA）转染或过表达载体操作，以了解特定基因对卫星细胞生理学的详细分子功能。凭借这些优势，可以通过免疫荧光染色在多个指定时间点分析卫星细胞的生理状态。在本方案中，我们提供了一种从趾长伸肌（EDL）肌肉中分离单根肌纤维、随后进行培养和免疫染色的高效实用方案。
