Skeletal Muscle Dissociation and FACS Isolation of FAPs

Details of skeletal muscle dissection have been previously described (13 (link)). In brief, muscle was dissected from Acvr1^{[R206H]FlEx/+}; Gt(ROSA26)Sor^CreERT2/+ mice and dissociated using the Skeletal Muscle Dissociation Kit (Miltenyi Biotec) and gentleMACS Octo Dissociator with heaters (Miltenyi Biotec), in accordance with the manufacturer’s instructions. Following centrifugation at 300g and 4°C for 10 minutes, the supernatant was discarded, and the pellet was resuspended in growth media (Dulbecco’s modified Eagle medium (DMEM; Life Technologies) with 50 U/mL penicillin, 50 μg/mL streptomycin (Gibco), and 16.6% fetal bovine serum (FBS; lot 192K18, Avantor). Cells were then plated onto tissue culture flasks (Corning). FACS was performed on single cells incubated with anti–mouse PDGFRA APC (clone APA5, eBioscience) to label FAPs, as previously described (13 (link), 35 (link)). FACS-isolated FAPs were seeded at a density of 2000 cells/cm² onto tissue culture flasks (Corning) in growth media and maintained at 37°C in a humidified atmosphere with 5% CO₂. Media were changed every other day. Prior to use, FAPs were treated with 2 μM (Z)-4-hydroxytamoxifen (Sigma-Aldrich) for 48 hours to induce inversion of the R206H-containing exon. All experiments were conducted with FAPs passaged fewer than 3 times.