Figure 4: In vitro modelling of anoxia and mitochondrial status of muscle stem cells.
From: Skeletal muscle stem cells adopt a dormant cell state post mortem and retain regenerative capacity
(a) Satellite cells were isolated by FACS from day 0, cultured at 4°C in normoxia or anoxia, then enumerated at indicated times (n=7 mice); (b, c) in vitro assessment of myotube formation and expression of myogenic markers Myogenin and Desmin with satellite cells cultured in anoxia (n=5 mice). (d) Measurement of ROS of cells that remain alive after 4 days in anoxia. Ratio between day 0, and 4 days in anoxia; (n=5 mice). (e) Fluorescence intensity quantification of TOM22 immunolabelling per cell in freshly isolated cells, in cells extracted 4 days post mortem and 4 days in anoxia (n=30 cells; n=4 mice per condition). (f–k) 3D imaging of mitochondrial mass in satellite cells (n=4 mice). (f, i) day 0; (g, j) 4 days post mortem; (h, k) 4 days post anoxia. Immunolabelling shown for mitochondria with TOM22 (red), and nuclei (blue) with Hoechst 33342. For each condition, side view and 3D volume rendering are shown; 360° view of these cells is shown in Supplementary Movies 6–8. (l) RT–qPCR of mitochondrial genes, 16S rRNA and CytB (n=6 mice). mt rRNA is present in greater amounts than mRNA, as expected58,59. (m) RT–qPCR of SOD1 and SOD2 (n=6 mice). (n) mtDNA content (12S region) estimation by qPCR (n=6 mice). Statistical analysis was performed using paired or unpaired Student's t-tests, a minimum of 95% confidence interval for significance; P-values indicated on figures are <0.05 (*), <0.01 (**), and <0.001 (***). Figures display average values of all tests ±s.e.m.
