EPC isolation and characterization

After ischemic muscle samples were collected, the bones were then separated for EPC isolation. The purified EPCs would be used directly for the following functional determination with no other treatment. The protocol of EPC isolation and culture was described previously [16, 17]. After the muscle sample was collected, the bones were then separated and smashed to collect bone marrow mononuclear cells (BM-MNCs). The bone sample includes hipbones, femurs, and tibiae, as well as shoulder bones, ulnas, vertebra, and sternum. EPCs were further isolated by layering the BM-MNCs on a density gradient (Histopaque 1083; Sigma) followed with centrifugation and were cultured in Endothelial Cell Basal Medium-2 (EBM-2; Lonza, Basel, Switzerland), supplemented with EGM-2 MV SingleQuots (Lonza). After 2 days of culture in a 37 °C, 5% CO₂ incubator, nonadherent cells were washed off by PBS while adherent cells were further incubated in fresh EBM-2 for 1 week before experiments.

Culture medium was gently washed off by PBS, and then cells were cultured in 5 μg/ml of DiI-Ac-LDL solution (Biomedical Technologies Inc., Stoughton, MA, USA) in EBM-2 for 4 hours at 37 °C, with the dish wrapped in aluminum foil. The cells were then fixed by 4% paraformaldehyde (PFA)/PBS for 20 min at 37 °C. After this, the cells were washed by PBS and DAPI staining solution (Beyotime Biotechnology, Shanghai, China) was added to stain the nuclei. After 10 min of incubation, staining solution were washed off by PBS and the sample was ready to be observed by fluorescent microscope.