Isolation of Fibroadipogenic Progenitors

PDGFR∝^Cre-Rosa26^Tdtomato mice (3 months old) were sacrificed with CO₂, and hindlimb muscles were harvested and digested as described above. After filtration and centrifugation, cell suspensions were then rinsed with 4 ml of FACS buffer (2% of heat inactivated FBS in HBSS). The samples were then divided into tubes considering the controls (unstained, single colors and fluorescence minus one). Cells were then centrifuged at 350×g for 5 minutes at 4°C and supernatant was discarded. Samples were incubated with corresponding antibodies diluted in FACS media (CD45- 2.5 μg/ml, CD31- 5 μg/ml and SCa-1- 0.5 μg/ml) for 30 minutes on a shaker plate. Then, 2–3 ml of FACS media was added to each tube and centrifuged (350×g minutes for 5 minutes at 4°C), supernatant was removed, and samples resuspended in FACS media. DAPI (1 μg/ml) was then added and samples were filtered through a 30 μm cell strainer and loaded into the Sony MA900 sorter. The gating strategy used to isolate Sca-1⁺ cells is shown on Supp. Fig. 1C–F. The negative (haematopoietic-CD45⁺ and endothelial-CD31⁺) and positive surface markers (stem cell antigen 1 or Sca-1⁺) were used to isolate FAPs,^{[64 (link)]} and FACS-isolated cells were confirmed to be TdTomato⁺.