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

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.¹.

Keywords: cell biology; cell culture; cell isolation; flow cytometry; microscopy; stem cells.

Graphical abstract

Figure 1

Photographs of proliferating myoblasts at 3 and 7 days post muscle stem cell extraction Scale bar, 100 μm.

Figure 2

PAX7 immunostaining of extracted myoblasts Scale bar, 100 μm.

Figure 3

Flow cytometry acquisition of myoblasts stained with Propidium Iodide and YO-PRO-1 (A) Gates obtained to observe the dual staining on samples, with the non-treated and H₂O₂ sample (positive control). (B) Percentage of early apoptotic cells observed in the positive control compared to Setdb1^+/+ and Setdb1^MuSC-KO condition. (C) Percentage of late apoptotic cells observed in the different conditions. We can observe an increase of late apoptotic cells in the Setdb1^MuSC-KO condition. Data are represented as mean ± SD. ∗p value < 0.05.

Figure 4

Flow cytometry acquisition of fixed myoblasts stained with EdU and Propidium Iodide (A) Gates obtained to observe the signal in the samples: SSC, FSC, PE, and APC. (B) Percentage of cells in G0/G1, S phase, and G2/M observed in the different Setdb1^+/+ and Setdb1^MuSC-KO condition. We can observe the decrease of cells in S phase in the Setdb1^MuSC-KO condition compare to Setdb1^+/+. However, there is no increase of G0-G1 and G2-M in the Setdb1^MuSC-KO. This can be explained by the cell death observed in the Setdb1^MuSC-KO condition. n = 3 biological samples. Data are represented as mean ± SD. ns, non-significant. ∗p value < 0.05.