Sca 1 pe cy7 clone d7

To compare the proliferative abilities of SCs in pharyngeal and hindlimb muscles in vivo, Bromo-2′-deoxyuridine (BrdU) assays were performed. Three-month-old male mice were injected with 10 µg BrdU (Sigma-Aldrich, St. Louis, MO)/g body weight intraperitoneally every 12 h for 2 days before sacrifice. Muscles were dissected and digested as described above. To assess proliferation, isolated mononucleated cells from pharyngeal or gastrocnemius muscles were immunostained with the following antibodies: 1:400 CD31-PE (clone 390; eBiosciences, San Diego, CA), 1:400 CD45-PE (clone 30-F11; BD Biosciences, San Jose, CA), 1:4000 Sca-1-PE-Cy7 (clone D7; BD Biosciences, Vancouver, Canada), and 1:20 a7-integrin-APC (FAB3518A; R&D SYSTEMS, Minneapolis, MN). Subsequently, cells were labeled for BrdU using a BrdU flow kit (BD biosciences, Vancouver, Canada), and proliferating SCs and FAPs were collected according to the following sorting criteria, CD31^-/CD45^-/Sca1^-/Intergrin7α⁺/BrdU⁺ and CD31^-/CD45^-/Sca1⁺/BrdU⁺, respectively using BD FACS LSR II or BD FACSymphony A3 flow cytometer (flow rate: 300–1,000 event/sec). Single cells were selected by FSC-A vs. FSC-H and then selected by SSC-A vs. SSC-H (Supplementary Figure S2A). Gating for SCs, FAPs, BrdU was drawn by FMO samples (Supplementary Figures S2B,C). Cells from gastrocnemius muscles were used as limb muscle controls.

To analyze the number of satellite cells and fibroadipose progenitor cells (FAPs) in injured muscles, muscles were dissected and digested with dispase II and collagenase II as previously described.^[53 (link)
^] Isolated mononucleated cells were immunostained with the following antibodies: 1:400 CD45‐PE (clone 30‐F11; BD Biosciences), 1:4000 Sca‐1‐PE‐Cy7 (clone D7, BD Biosciences), 1:400 CD31‐PE (clone 390; eBiosciences). Fibroadipose progenitor cells were counted using the following criteria: CD31⁻/CD45⁻/Sca1⁺ and satellite cells are counted by tdTomato⁺ using BD LSR II cytometry analyzer and analyzed using FCS Expression 6 Flow software 6.01.