Vegfr2 kdr pe

ECFC single-cell suspension was generated by detaching cells with TrypLE™ Express Enzyme (Gibco, USA) and resuspended to a concentration of 1 × 10⁷ cells/ml. Samples were incubated, respectively, with anti-human CD31-FITC (eBioscience, USA), VEGFR2/KDR-PE (R&D, USA), CD144-FITC (Abcam, UK), CD34-PE (Biolegend, USA), CD45-FITC (Biolegend, USA), and CD14-FITC (eBioscience, USA). MSCs were resuspended to a concentration of 1 × 10⁶ cells/ml and incubated, respectively, with anti-human CD29-PE (Biolegend, USA), CD90-PE (Biolegend, USA), CD14-FITC (Biolegend, USA), CD19-PE (Biolegend, USA), CD73-FITC (Biolegend, USA), CD105-FITC (Biolegend, USA), HLA-DR-PE (Biolegend, USA), CD34-PE (Biolegend, USA), CD45-FITC (Biolegend, USA), and CD31-FITC (eBiosciences, USA). 5 μl antibody solution was added into 100 μl cell suspension and incubated for 30 minutes at 4°C in the dark; 400 μl of PBS was added and cells were analyzed with FACSAria I (Becton Dickinson, USA) or Accuri C6 (Becton Dickinson, USA) and Becton Dickinson CELLQuest software.

Flow cytometric detection of CEC and CEP were carried out at the same day of blood sampling according to the protocol published by Duda et al. [44 (link)]. In brief, PBMC isolation was performed with FICOLL density gradient centrifugation and afterwards FC blocking was performed (Supplementary Figure 2). PBMC were incubated in triplicates with antibodies specific for CD31- FITC (BD Pharmigen), CD45- PerCP (BD Pharmigen), CD133- PE (MiltenyiBiotec), CD34- PC7 (Beckman Coulter), VEGFR2 (KDR)- PE (R & D Systems) and CD146- PE (BD Pharmigen). Appropriate fluorchrome-conjugated isotype-matched murine IgG antibodies (BD Pharmigen) were used as controls for each staining procedure. After incubation for 30 minutes at 4°C, cells were washed, resuspended and analyzed in a Cytomics-FC-500 cytometer using the Cytomics RXP-Software. CEC were defined as CD45⁻/CD31⁺/CD146⁺ cells and CEP were defined as CD45⁻/CD31⁺/CD133⁺ cells. Further we determined CD45⁻/CD31⁺/VEGFR2⁺ cells (cell gating and CEC phenotype definition depicted in Supplementary Figure 3).