FACS-based Satellite Cell Isolation From Mouse Hind Limb Muscles.
FACS-based Satellite Cell Isolation From Mouse Hind Limb Muscles.
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DOI：
10.21769/bioprotoc.1558
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发表时间：
2015-08
期刊：
影响因子：
0.8
通讯作者：
A. Gromova;Matthew T. Tierney;A. Sacco
中科院分区：
文献类型：
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作者：
A. Gromova;Matthew T. Tierney;A. Sacco
荧光激活细胞分选(FACS)是从成年小鼠骨骼肌中分离纯化卫星细胞或肌肉干细胞的灵敏而准确的方法(Liu等人，2013年；Saco等人，2008年；Tierney等人，2014年)。后肢肌肉的机械和酶消化使单核肌肉细胞进入悬浮状态。该方案采用分离策略，通过磁分离和基于FACS的排除来耗尽表达细胞表面标志CD45、CD31、CD11b和Ly-6A/E-Sca1的细胞，并积极选择表达A7-整合素和CD34的细胞。这使得研究人员能够成功地丰富卫星细胞，这些卫星细胞统一表达配对框转录因子Pax7，并能够长期自我更新、骨骼肌修复和肌肉干细胞库重新繁殖。
Fluorescence Activated Cell Sorting (FACS) is a sensitive and accurate method for purifying satellite cells, or muscle stem cells, from adult mouse skeletal muscle (Liu et al., 2013; Sacco et al., 2008; Tierney et al., 2014). Mechanical and enzymatic digestion of hind limb muscles releases mononuclear muscle cells into suspension. This protocol employs fractionation strategies to deplete cells expressing the cell surface markers CD45, CD31, CD11b and Ly-6A/E-Sca1, both by magnetic separation and FACS-based exclusion, and positively select for cells expressing a7-integrin and CD34. This enables the researcher to successfully enrich satellite cells that uniformly express the paired-box transcription factor Pax7 and are capable of long-term self-renewal, skeletal muscle repair and muscle stem cell pool repopulation.