Isolation and Sorting of Fibro-Adipogenic Progenitors

FAPs were isolated by FACS as described^{34 (link)}, with minor modifications, as follows. Total hindlimb muscle was harvested, minced with scissors for 7–9 min and then incubated for 60–75 min at 37 °C in Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies) supplemented with 700–800 U/ml Collagenase Type II (Worthington Biochemical) and 0.3 U/ml Dispase (Invitrogen) with gentle agitation every 15 min. Enzymatic digestion was then quenched by addition of DMEM containing 20% HyClone™ characterized fetal bovine serum (FBS) (GE Healthcare; Lot# A00168), followed by serial filtration of the cell suspension through 100 µm and 70-µm cell strainers (Falcon). The cell suspension was then centrifuged at 800×g for 5 min and resuspended in 10% FBS in 1× Dulbecco’s phosphate-buffered saline (DPBS) (Gibco) for antibody staining. Anti-CD31 (1:100, Miltenyi Biotech, #130-097-418) and anti-CD45 (1:100, Miltenyi Biotech, #130-052-301) microbead conjugated antibodies were used to deplete endothelial and hematopoietic cells, respectively, via MACS LS Separation columns (Miltenyi Biotech) loaded in a QuadroMACS Separator (Miltenyi Biotech), according to manufacturer’s instructions. Following magnetic depletion, the remaining cell suspension was incubated with fluorescently conjugated SCA-1-V450 (1:400, BD Horizon, #560653) and PDGFRα-APC (1:100, eBioscience, #17-1401-81) antibodies for 30 min on ice, centrifuged at 800×g for 5 min, and resuspended in 2% FBS in DPBS following two washes with 1× DPBS. Samples were filtered through 35-µm cell strainers (Falcon) and 50 µg/mL 7-AAD (BioLegend) was added to a final concentration of 0.50 µg/mL immediately prior to sorting to distinquish between live/dead cells. Fluorescence-minus-one (FMO) controls were used to test for non-specific staining and generate strict sort gates to minimize cross contamination between populations. FAPs were isolated based on co-staining for PDGFRα and SCA-1 as shown in Supplementary Fig. 2. To distinguish between Acvr1^R206H/+ and wild-type FAPs, we assessed the enriched hindlimb PDGFRα+ SCA1+ subpopulation for tdTomato and GFP expression, which provides a direct readout of recombination status at the Acvr1^R206H and R26^NG loci, respectively. For the tdTomato-FMOs, depending on the experimental mouse line being analyzed, either R26^NG/+;Tie2-Cre or R26^NG/+;Pdgfrα-Cre mice were utilized. Acvr1^tnR206H/+ mice were used for the GFP FMOs. Sorting and analysis was done on a FACS Aria II (BD Biosciences) equipped with 407, 488, and 633 lasers. Acvr1^R206H and R26^NG gates were confirmed based on verification of tdTomato and GFP expression via fluorescence microscopy.