Fitc uea 1

EPCs were washed with phosphate-buffered saline (PBS) three times, and incubated in medium containing 20 μg/ml Dil-ac-LDL (YEASEN Biotech, China) for 4 h at 37 °C, 5% CO₂. Cells were fixed with 4% paraformaldehyde and incubated for 1 h with 10 μg/ml FITC-UEA-1 (Invitrogen, USA) at room temperature. Nuclei were labeled with DAPI (C0065, Solarbio, China). The incorporation of Dil-ac-LDL and binding of FITC-UEA-1 were assessed and photos were taken in an Intelligent Full-Automatic Fluorescence Microscopy Imaging System (Invitrogen™ EVOS™ FL Auto 2, Thermo Scientific, USA).

Second-generation DB/+-EPCs and DB/DB-EPCs were incubated at room temperature with 10 μg/ml Dil-Ac-LDL (Biomedical Technologies, Stoughton, MA) for 4 h and then fixed in 4% paraformaldehyde for 15 min. The fixed cells were washed twice in PBS (pH 7.4). Subsequently, cells were incubated for 1 h with 10 μg/ml FITC-UEA-1 (Molecular Probes, USA). The images of fluorescently labeled cells were captured using a laser scanning confocal microscope (Leica, Germany). Cells positive for both markers are considered EPCs [30 (link)].

For flow cytometry, EPCs on day 7 were detached and resuspended in PBS. After Fc-blocking, 1.0 × 10⁶ cells were incubated with conjugated anti-CD133-PE (BD Biosciences, San Jose, CA) and CD34-FITC (BD Biosciences, San Jose, CA) for 20 min at 4 °C. Cells were analyzed using Beckman Coulter FC500 flow cytometer. To observe DiI-ac-LDL uptake and FITC-UEA-1 binding, EPCs were incubated with 2.4 μg/mL Dil-Ac-LDL (Invitrogen-Molecular Probes, Eugene, OR) in EGM-2 medium at 37 °C for 4 h. Then, cells were fixed in 4% paraformaldehyde and further incubated with 20 μg/mL FITC-UEA-1 (Introvogen-Molecular Probes, Eugene, OR) for 1 h. For immunofluorescence staining, EPCs were fixed in 4% paraformaldehyde for 10 min, blocked in 5% normal goat serum, and then incubated with anti-CD133-PE (BD Biosciences, San Jose, CA) and anti-CD34-FITC (BD Biosciences, San Jose, CA) antibodies at 4 °C overnight. Fluorescence was observed under a confocal laser scanning microscope.