Anti human cd31 fitc

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.

On days 1, 4, and 7 of direct cell co-culture with CGF, the medium was discarded, and 0.1% type IV collagenase (Gibco) was added. This was then digested in the incubator with 5% CO₂ at 37°C for 30 min. After another rinse in phosphate-buffered saline (PBS), 0.25% trypsin was added and digested again at 37°C for 1–2 min to completely disperse the cells. Complete medium (3–4 times the volume of trypsin) was added to terminate digestion. After this, single cell suspensions were collected and centrifuged at 1,000 rpm for 5 min. The cryosupernatant was discarded, PBS was added to resuspend cells, and the total number of cells was counted. This was followed by another centrifugation and the addition of 100 μL PBS and 5 μL anti-human CD31 FITC (eBioscience, San Diego, CA, United States). After incubation on ice for 30 min in the dark, 500 μL PBS was added to resuspend to single cell suspension. Flow cytometry was used to count the proportion of different cells. CD31-positive cells were defined as HUVECs and CD31 negative cells as hBMSCs. The proportions of positive and negative cells were multiplied respectively by the total number of cells to quantify the numbers of HUVECs and hBMSCs in each group.