MuSCs were FACS-sorted from hind limb muscles in postnatal 1-week mice according to the reported protocol [45 (link)]. Briefly, mechanical and enzymatic dissociation of cells, released resident mononucleated cells, then followed by antibody staining and FACS isolation.
Mononuclear cells were incubated with antibodies against CD45 (PE/Cy7-CD45, eBioscience, 25-0451-82), CD31 (FITC-CD31, BioLegend, 102506), Sca1 (V450-Sca1, BD, 560653) and VCAM1 (efluor660-VCAM1, eBioscience, 50-1061-80). Immunostained cells were briefly washed, passed through a 70-μM nylon mesh (Falcon) and suspended at a concentration of 106–107 cells per mL. Cells were further separated with the Beckman Coulter MoFlo XDP system. Sorting gates were strictly defined on the basis of control cells stained with single antibodies as well as the patterns of forward scatter and side scatter of MuSCs in preliminary tests. Collection of the cells that were positive for VCAM1 expression and negative for CD31, CD45, and Sca1.
Mononuclear cells were incubated with antibodies against CD45 (PE/Cy7-CD45, eBioscience, 25-0451-82), CD31 (FITC-CD31, BioLegend, 102506), Sca1 (V450-Sca1, BD, 560653) and VCAM1 (efluor660-VCAM1, eBioscience, 50-1061-80). Immunostained cells were briefly washed, passed through a 70-μM nylon mesh (Falcon) and suspended at a concentration of 106–107 cells per mL. Cells were further separated with the Beckman Coulter MoFlo XDP system. Sorting gates were strictly defined on the basis of control cells stained with single antibodies as well as the patterns of forward scatter and side scatter of MuSCs in preliminary tests. Collection of the cells that were positive for VCAM1 expression and negative for CD31, CD45, and Sca1.