Immunofluorescence analysis of myogenic differentiation.
Immunofluorescence analysis of myogenic differentiation.
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DOI：
10.1016/bs.mcb.2022.02.010
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发表时间：
2022
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骨骼肌是一种高度再生的组织，可以有效地从受伤和过度运动造成的各种损伤中恢复。在成人肌肉中，称为卫星细胞的干细胞在有丝分裂时静止，但在肌肉损伤时被激活，作为肌源性前体细胞或成肌细胞进入细胞周期。经过几轮细胞周期后，它们存在于细胞周期中，并相互融合形成多核肌管，最终成熟成为收缩性肌纤维。卫星细胞可以很容易地从小鼠骨骼肌分离酶消化和磁性分离与特异性表面标记的抗体。C2C12细胞是一种永生化的小鼠成肌细胞系，其可商购获得并且比原代成肌细胞更容易扩增。原代成肌细胞和C2C12细胞已被广泛用作肌源性分化的体外模型。对这一过程的适当检查需要监测亚细胞区室中的特异性蛋白质表达，这可以通过免疫荧光染色来完成。本章描述了从小鼠骨骼肌中分离卫星细胞的工作流程，随后进行免疫荧光染色以评估原代成肌细胞和C2C12细胞的增殖和分化。
Skeletal muscle is a highly regenerative tissue that can efficiently recover from various damages caused by injuries and excessive exercises. In adult muscle, stem cells termed satellite cells are mitotically quiescent but activated upon muscle damages to enter the cell cycle as myogenic precursor cells or myoblasts. After several rounds of cell cycles, they exist the cycle and fuse to each other to form multinucleated myotubes, and eventually mature to become contractile myofibers. Satellite cells can be readily isolated from mouse skeletal muscle with enzymatic digestion and magnetic separation with antibodies against specific surface markers. C2C12 cells are an immortalized mouse myoblast cell line that is commercially available and more readily expandable than primary myoblasts. Both primary myoblasts and C2C12 cells have been extensively used as useful in vitro models for myogenic differentiation. Proper examination of this process requires monitoring specific protein expression in subcellular compartments, which can be accomplished through immunofluorescence staining. This chapter describes the workflow for the isolation of satellite cells from mouse skeletal muscle and subsequent immunofluorescence staining to assess the proliferation and differentiation of primary myoblasts and C2C12 cells.