Cell isolation was performed as described previously (48). Minced muscles were digested with 0.2% type II collagenase (Worthington) for 60 minutes (30 minutes for human muscles) at 37°C. Digested muscles were passed through an 18-G needle several times and further digested for 15 minutes at 37°C. Muscle slurries were filtered through a 100 μm cell strainer and then through a 40 μm cell strainer (BD Biosciences). Mouse cells were resuspended in a washing buffer consisting of 2.5% FBS in PBS and stained for cell sorting. Human cells were cultured on a collagen I–coated dish (Iwaki) in growth medium (GM) consisting of DMEM supplemented with 20% FBS, 1% penicillin-streptomycin, and 2.5 ng/mL bFGF (Katayama Chemical) at 37°C under 5% CO2 and 3% O2 for several days. When cells reached 80% confluence they were trypsinized and stained with primary antibodies followed by secondary staining.

The atrium was removed, and each ventricle was finely minced. Minced hearts were digested with 0.2% type II collagenase (Worthington) for 5 minutes at 37°C. Digested hearts were passed through an 18-G needle several times. Digestion and needle homogenization were repeated 3 times. Slurries were filtered through a 100 μm cell strainer and then through a 40 μm cell strainer (BD Biosciences). Cells were resuspended in washing buffer and stained for cell sorting.

Bone marrow cells were flushed out from the femurs and tibias and pooled in sterile tubes. The remaining femurs and tibias were cut into small pieces. Bone fragments were digested with 0.2% type II collagenase (Worthington) with gentle agitation for 2 hours at 37°C. Digested bone fragments were filtered through a 100 μm cell strainer and then through a 40 μm cell strainer (BD Biosciences). Filtered cells were mixed with pooled bone marrow cells. Cells were resuspended in washing buffer and stained for cell sorting.

Reagents used for cell surface staining are listed in Supplemental Table 2. Isotype control staining is presented in Supplemental Figure 6. Cell sorting was performed with the MoFlo Astrios (Beckman Coulter) or SORP FACSAria II (BD Biosciences) instrument.

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