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使用流式细胞术对原代肌肉干细胞培养物进行细胞增殖和细胞死亡分析的实验方案。

Protocol for cell proliferation and cell death analysis of primary muscle stem cell culture using flow cytometry.

作者信息

Garcia Pauline, Mercier Orane, Ravent Jade, Le Grand Fabien

机构信息

Université Claude Bernard Lyon 1, CNRS UMR 5261, INSERM U1315, Institut NeuroMyoGène, Pathophysiology and Genetics of Neuron and Muscle Unit, 69008 Lyon, France.

Université Claude Bernard Lyon 1, CNRS UMR 5261, INSERM U1315, Institut NeuroMyoGène, Pathophysiology and Genetics of Neuron and Muscle Unit, 69008 Lyon, France.

出版信息

STAR Protoc. 2024 Dec 20;5(4):103411. doi: 10.1016/j.xpro.2024.103411. Epub 2024 Oct 23.

Abstract

Skeletal muscle is critically dependent on the function of muscle stem cells (MuSCs) for effective muscle repair following injury. Here, we detail a protocol for the isolation of primary muscle cells and subsequent analysis of proliferation capacity in vitro using EdU (5-ethynyl-2'-deoxyuridine) on fixed cells. We also describe a cell death analysis on living cells with the identification of early- and late-apoptotic cells, as well as necrotic cells, through the incorporation of propidium iodide and YO-PRO-1 staining. For complete details on the use and execution of this protocol, please refer to Garcia et al..

摘要

骨骼肌严重依赖肌肉干细胞(MuSCs)的功能来在损伤后进行有效的肌肉修复。在此,我们详细介绍一种分离原代肌肉细胞的方案,以及随后使用EdU(5-乙炔基-2'-脱氧尿苷)对固定细胞进行体外增殖能力分析的方法。我们还描述了一种对活细胞进行细胞死亡分析的方法,通过碘化丙啶和YO-PRO-1染色来鉴定早期和晚期凋亡细胞以及坏死细胞。有关此方案的使用和执行的完整详细信息,请参考加西亚等人的研究。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ba06/11539148/57e62c3bee46/fx1.jpg

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