Cd45 pe cy7

For identification of fibroblast population, CD31-FITC (4,220,516, BD Biosciences, USA), CD45-PE/Cy7 (561,868, BD Biosciences, USA), CD326 (EPCAM)-PE (2,088,498, Invitrogen, USA) were used to distinguish fibroblast from T cells, endothelial cells and epithelial cells. PDGFRα (3174, CST) and α-SMA (ab32575, Abcam) were used to distinguish iCAF from myCAF.

Hind limb muscles were dissected, finely minced and dissociated by digestion with Collagenase II (0.2%) in Ham’s F10 medium containing 10% house serum for 90 min. Then the fragmented myofibers were washed and further digested in Collagenase II (0.2%) and dispase (2.5 units per ml; Invitrogen) for an additional 30 min. Digested fiber suspensions were triturated to yield a mononuclear cell suspension. Mononuclear cells were stained with Vcam-biotin (BD Bioscience, 1:50, clone 429), Streptavidin-APC was used to amplify the Vcam Signal (BD Bioscience, 1:75, clone 17-4317-82), Sca1-FITC (BD Bioscience, 1:100, clone D7), CD31-PE-cy7 (BD Bioscience, 1:100, clone 390) and CD45-PE-Cy7 (BD Bioscience, 1:100, clone 30-F11). Fluorescence-activated cell sorting (FACS) was performed on the stained cells using a BD FACSAria II cell sorter to isolate CD31⁻/CD45⁻/Sca1⁻/Vcam⁺ satellite cells using a previously described gating strategy^{32 (link)} and detailed in Supplementary Fig. 4. For satellite cell culture experiments, sorted satellite cells were plated on Matrigel (Sigma, 1:100)-coated dishes and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 20% fetal bovine serum (FBS), 10% horse serum (HS) and 3% chicken embryo extract (CEE). To differentiate satellite cells, cells were cultured in DMEM with 2% HS.

To obtain purified SCs, primary cells were isolated as described previously (Paris et al., 2016 (link)). Muscles were dissected from mice and digested using collagenase II (Gibco, Carlsbad, California) in Dulbecco's modified Eagles medium (DMEM, Sigma-Aldrich) for 60 min at 37°C with agitation. The suspension was then washed and digested in collagenase II and dispase (Gibco) for 30 min at 37°C with agitation. The resultant mononuclear cells were then stained with the following antibodies: CD31-PECy7 (BD Biosciences 561410, San Jose, California), CD45-PECy7 (BD Biosciences 552848), Sca1-FITC (BD Biosciences 562058), Integrin α7 (ITGA7)-Alexa Fluor 647 (AbLab, Vancouver, Canada) and VCAM-PE (Invitrogen RMCD10604). FACS was performed using a FACSAria II Cell Sorter (BD Biosciences) and SCs were collected according to the following sorting criteria: CD31-/CD45-/Sca1-/ITGA7+/VCAM+. FACs-purified SCs were plated at 3000 cells per well in eight-well Permanox chamber slides (Nunc Lab-Tek, Carlsbad, California) and cultured for five days in DMEM with 10% Horse Serum (Thermo Fisher Scientific, Carlsbad, California) and 5 ng/mL FGF2 (Cell Signaling Technologies, Danvers, Massachusetts).

Fluorescein-conjugated ShK-F6CA was purchased from Peptides International (Louisville, KY) [17 (link), 18 (link)]. Fluorophore-conjugated antibodies for flow cytometry were obtained from BD Biosciences (CD11b-APC-Cy7, CD45-PE-Cy7, CD45-FITC, Ly6c-PE) [13 (link)]. Polystyrene phycoerythrin-fluorescent 1-μm microspheres (Thermo Fisher #F13083) were used for phagocytosis assays [16 (link)]. Percoll was purchased from Sigma-Aldrich (#P1644).

The phenotype of NPCs was determined by cytofluorimetric analysis with fluorochrome‐conjugated antibodies against human CD90‐FITC (#555595) at 1:100, CD73‐PE (#550257) at 1:100, CD105‐Alexa488 (#MHCD10520) at 1:100, CD34‐PECy5 (#555823) at 1:100, CD45‐PECy7 (#557748) at 1:100, CD146‐BV605 (#7433019) at 1:100, and Tie2‐PE (#FAB3131A) at 1:50 (all from BD Bioscience, Franklin Lakes, NJ). Isotype IgG was used as a control (all from BD Biosciences). Cells in suspension were incubated for 40 minutes with each of these antibodies at 4°C in FACS buffer (PBS, 0.5% human serum albumin, 0.5 mm EDTA), and analyzed with a Cell Lab Quanta SC flow cytometer (Beckman Coulter Inc.).