University Health Network, Toronto, ON, Canada.
Department of Biology, Trent University, Peterborough, ON, Canada.
Methods Mol Biol. 2025;2857:169-180. doi: 10.1007/978-1-0716-4128-6_16.
Acute skeletal muscle injury initiates a process of necrosis, debris clearance, and ultimately tissue regeneration via myogenesis. While skeletal muscle stem cells (MuSCs) are responsible for populating the proliferative myogenic progenitor pool to fuel muscle repair, recruited and resident immune cells have a central role in the regulation of muscle regeneration via the execution of phagocytosis and release of soluble factors that act directly on MuSCs to regulate myogenic differentiation. Therefore, the timing of MuSC proliferation and differentiation is closely linked to the populations and behaviors of immune cells present within skeletal muscle. This has important implications for aging and muscle repair, as systemic changes in immune system function contribute to a decline in muscle regenerative capacity. Here, we present adapted protocols for the isolation of mononuclear cells from skeletal muscles for the quantification of immune cell populations using flow cytometry. We also describe a cardiotoxin skeletal muscle injury protocol and detail the expected outcomes including immune cell infiltration to the injured sites and formation of new myocytes. As immune cell function is substantially influenced by aging, we extend these approaches and outcomes to aged mice.
急性骨骼肌损伤会引发坏死、碎片清除过程，最终通过成肌作用实现组织再生。虽然骨骼肌干细胞（MuSCs）负责填充增殖性成肌祖细胞池，以促进肌肉修复，但募集和驻留的免疫细胞通过执行吞噬作用并释放直接作用于 MuSCs 以调节成肌分化的可溶性因子，在调节肌肉再生方面发挥着核心作用。因此，MuSC 的增殖和分化时间与骨骼肌中存在的免疫细胞的群体和行为密切相关。这对衰老和肌肉修复具有重要意义，因为免疫系统功能的全身性变化会导致肌肉再生能力下降。在这里，我们提供了从骨骼肌中分离单核细胞用于使用流式细胞术定量免疫细胞群体的改良方案。我们还描述了一种心脏毒素骨骼肌损伤方案，并详细介绍了预期结果，包括免疫细胞浸润到损伤部位和新肌细胞的形成。由于免疫细胞功能受衰老的影响很大，我们将这些方法和结果扩展到老年小鼠。
