Sca 1 fitc

Mice were sacrificed by exsanguination via cardiac puncture and lungs were perfused clear of blood with saline and removed from the thoracic cavity, as previously described [25] . Lung tissue-associated cells were extracted from the right lung by mincing and enzymatic digestion, as previously described [20] (link). Mononuclear cells were collected following density gradient centrifugation (400 g for 20 min) over Histopaque (Sigma, Oakville, Ontario, Canada).
Cells were immunostained with Sca-1-FITC, c-kit-PE (BD Bioscience, Oakville, ON, Canada) and VEGFR2-APC (eBioscience Inc., San Diego, CA, USA), or isotype control antibodies (40 min at 4°C), and fixed in PBS with 1% paraformaldehyde (BDH Laboratory Supplies, Mississauga, ON, Canada), and cell data (100,000 events in the lymphomononuclear region) were acquired using a FACSCaliber flow cytometer equipped with a 488-nm argon ion laser (BD Instrument Systems, Mississauga, ON, Canada). Primitive progenitor cells (Sca-1⁺c-kit⁺) and lineage-committed EPCs (Sca-1⁺c-kit⁺VEGFR2⁺) were enumerated using the Cellquest software package (BD Biosciences). The flow cytometric gating strategy are previously described in detail in [15] (link). Doyle et al., 2011 [20] (link). Absolute numbers of cells were calculated using the percentage of population positivity obtained by flow cytometery and the total white cell count.

Recombinant human thrombopoietin (rhTPO), interleukin I-beta (rhIL-1β), interleukin 6 (rhIL-6), stem cell factor (rhSCF), nerve growth factor beta (rhNGF-β), and granulocyte-macrophage colony stimulating factor (rhGM-CSF) were purchased from R&D systems (Minneapolis, MN, USA). K252a was purchased from Calbiochem (San Diego, CA, USA). The following fluorochrome-conjugated anti-human antibodies were used for flow cytometry analysis: FITC-labelled human lineage cocktail 4 (CD2, CD3, CD4, CD7, CD8, CD10, CD11b, CD14, CD19, CD20, CD56, CD235a), Sca-1-FITC, CD34-PE Cy7, CD41-APC, TrkA-PE (all from BD Pharmingen, San Diego, CA, USA), CD61-AF 647 were obtained from Biolegend (San Diego, CA, USA). The nuclear dyes, 7-Aminoactinomycin D (7-AAD) and propidium iodine (PI), were also from BD Pharmingen.

Single cell suspension dissociated from S1M allografts and primary mouse cells were stained with Sca‐1 FITC (553335; BD Pharmingen), CD133 PE (12‐1331‐82; eBioScience), CD44 FITC (11‐0441‐81; eBioScience), CD45 PE‐CyTM7 (552848; BD Pharmingen), rat IgG isotype control PE (553930; BD Pharmingen), and rat IgG isotype control FITC (11‐4031‐81; eBioScience) for 1 h at 4°C in the dark room. The cells were analyzed on FACS Calibur (BD Biosciences) and sorted on an Aria cell sorter (BD Biosciences).

Hind limb muscles from C57/BL6 wild type mice (Charles River Laboratories) were minced and digested in PBS (Sigma) containing 0.1% BSA, 300 µg/ml Collagenase A (Roche), 0.24 U/ml Dispase I (Roche), 2 μg/ml DNase I (Roche), 50 μM CaCl2 and 1 mM MgCl2 for 60 min at 37 °C under constant agitation. For fluorescence-activated cell sorting, digested muscle cells were stained with primary antibodies (1:50) CD3-eFluor450 (eBioscience), CD45-eFluor450 (eBioscience), Ter119-eFluor450 (eBioscience), Sca-1-FITC (BD Pharmingen), and α7integrin-APC (AbLab) for 30 min at RT. Cells were finally washed and resuspended in Running Buffer (PBS, 0.1% Sodium Azide, 0.2% FBS). Flow cytometry analysis and cell sorting were performed on a DAKO-Cytomation MoFlo High Speed Sorter.
Muscle satellite cells (SCs) were isolated as Ter119−/CD45−/CD31−/α7-integrin+/Sca-1− cells. Fibro-adipogenic progenitors (FAPs cells) were isolated as Ter119−/CD45−/CD31−/α7-integrin−/Sca-1+ cells, as previously described^{44 (link)}.

Satellite cells were isolated from skeletal muscles of 4-week-old mice by FACS, after enzymatic dissociation with a solution of CMF containing 0.02% BSA (Bovine Serum Albumin) (Sigma-Aldrich), 1% of Penicillin/Streptomycin (Sigma-Aldrich), 0.25 mg/ml Dispase II (Roche); 2 μg/μl Collagenase A (Roche), 8 mM CaCl₂,10 ng/μl DNase I (Roche) and 5 mM MgCl₂ for 2 hours at 37 °C. Cell suspensions were filtered through a 100-μm and a 40-μm nylon filters (Falcon) and stained with primary antibodies for 30 min on ice. The following antibodies were used: CD45- eFluor450 (eBioscience), Ter119- eFluor450 (eBioscience), CD31- Pacific Blue (Invitrogen) and Sca1-FITC (BD Bioscience), integrin- α7–APC (Ab-Lab). Flow cytometry analysis and cell sorting were performed on a DAKO-Cytomation MoFlo High Speed Sorter.