Fluorescence activated cell sorting (FACS) and FAP expansion

Details of skeletal muscle dissection and isolation of FAPs have been previously described (Biswas and Goldhamer, 2016; Lees-Shepard et al., 2018; Wosczyna et al., 2012). Sorting was performed on a FACS Aria II (BD Biosciences, Franklin Lakes, NJ) equipped with 407, 488, and 633 lasers. FACS-isolated FAPs were seeded at a density of 2000 cells/cm² onto tissue culture flasks (Nunc, Rochester, NY) in Dulbecco's Modified Eagle Medium (DMEM; Life Technologies, Carlsbad, CA) with 50 U/mL Penicillin and 50 μg/mL Streptomycin (Pen/Strep; Gibco, Billings, MT) and 20% HyClone fetal bovine serum (FBS), characterized (GE Healthcare, Chicago, IL; Lot# A00168). FAPs were maintained at 37°C in a humidified atmosphere at 5% CO₂. Media was changed every other day. All experiments utilized FAPs passaged fewer than three times.