Isolation of CD34+ VEGFR-2+ Cells

After overnight fasting, 5 mL peripheral blood samples were collected in BD Vacutainer® CPT™ Tubes (USA) from the subjects. The tubes were gently mixed upside down 10 times and stored vertically. Within two hours, the tubes were centrifugated at 1500 g, 20 min, and 25 °C. The mononuclear precursor cells were isolated, washed, and resuspended in PBS (Gibco, USA). The isolated cells were incubated with two kinds of monoclonal antibodies: PE-conjugated anti-human CD34 (BD, USA) and allophycocyanin-conjugated anti-human VEGFR-2 (R&D Systems, USA). The cell sorting was performed on a FACSAria III (BD Biosciences, USA) at 488 nm to obtain double-positive cells, CD34 + VEGFR-2 + cells [23 (link)].