Mouse satellite cell isolation, cloning and <i>in vitro</i> proliferation.

<p>(A) Experimental plan<b>.</b> For satellite cell (SC) isolation, hindlimb skeletal muscles of C57BL/6J mice were digested and single myofibers collected. SCs were mechanically disengaged (stripping passage) and cloned with the limited dilution method. For each experiment, cloned cells were randomly cultured in normoxic (20% O₂) or hypoxic (2% O₂) conditions with a proliferative medium. (B) After 5 days of culture, all the clones had a similar morphology (bar = 75 µm). (C) The number of clones generated in hypoxia was higher than in the normoxia conditions, expressed as the number of clones obtained starting from the same seeded cell number (n = 16, mean±SEM, * p<0.05). (D and H) Number of cells <i>per</i> clone distribution (example experiment) under normoxia or hypoxia conditions respectively (a model based cluster analysis with normal mixture modelling has been applied to identify the groups): cells distribute in 2 clusters easily separable with a cut-off value (light grey bar) in LPC and HPC. (E and I) Averages of the number of cells <i>per</i> clone both in LPC and HPC demonstrated that the proliferation increases in those clones cultured with 2% O₂ (total number of clones counted: 20% O₂ n = 600, 2% O₂ n = 700, mean±SEM, ** p<0.01, *** p<0.001). (F and L) Pie charts indicate LPC and HPC proportions calculated as percentage of the total number of clones (n = 16, mean±s.d., 20% O₂: LPC = 73%±8.1, HPC = 27%±8.1; 2% O₂: LPC = 69%±8.3, HPC = 31%±8.3; p<0.001). (G and M) ATP quantification: LPC show lower ATP content with respect to HPC, both in normoxia and hypoxia. ATP measurements uphold the higher number of cells both in LPC and HPC under hypoxia (n = 132, mean±SEM, *** p<0.001).