Isolation and Characterization of Mouse Bone Marrow-Derived Endothelial Progenitor Cells

EPCs are derived from mouse bone marrow, as previously described^{27 (link)}. Simply, mononuclear cells were isolated from bone marrow, inoculated in a culture bottle, incubated with EGM-2 (Lonza, Switzerland), and placed in an atmosphere at 37°C, 95% humidity and 5% CO₂. On the 4th day of incubation, we replaced the old medium with fresh medium, and removed the cells that became unattached to the wall. Every three days, we removed half of the old medium and replace it with a fresh one. Cells were collected on the 7th day of culture.
Three methods were applied to identify the EPCs^{27 (link)}: morphology of EPCs, double staining with 1,1’-dioctadecyl-3,3,3’,3-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (Dil-acLDL from Eugene, USA) and fluorescein isothiocyanate-labeled Ulex europaeus agglutinin-1 (FITC-UEA-1 from Sigma, USA), and co-expressing of FITC-CD34 + / PE-CD133 + / APC-Flk-1 + (FITC-CD34 and APC-Flk-1 from Becton Dickinson, USA; PE-CD133 from eBioscience, USA). PerCP-conjugated anti-mouse Sca-1 antibody (PerCP-Sca-1 from Biolegend, USA) was used to detect Sca-1 positive rate.