Pe labeled monoclonal mouse anti human antibodies recognizing cd31

Late EPCs were cultured and characterized as in our previous study [20 (link)]. Briefly, PBMCs from healthy subjects were cultured on fibronectin-coated 6-well plates in EBM-2 supplemented with endothelial growth medium-SingleQuots (Clonetics, San Diego, CA, USA). After a 4-day culture, non-adherent cells were removed by thoroughly washing with culture medium. Medium was changed daily for 7 days, and then every other day until the first passage. For all assays, LEPCs were used at passages 3 (about 28 days). After 28 days of culture, marker proteins of cultured EPCs were examined by flow cytometry analysis using phycoerythrin (PE)-labeled monoclonal mouse anti-human antibodies recognizing CD31 (BD Pharmingen), Tie-2 receptor (BD Pharmingen) and kinase-insert domain receptor (KDR) (R&D system). Overall, (92.25 ± 4.8)% of the cells were positive for CD31, (80.16 ± 6.5)% for KDR, and (88.36 ± 5.6)% for Tie-2 receptor. Based on the isolation and cultivation protocol, the adherent mononuclear cells were identified as late EPCs.

The expression of endothelial marker proteins was examined in the cultured EPCs by using flow cytometric analysis with phycoerythrin (PE)-labeled monoclonal mouse anti-human antibodies recognizing CD31 (BD Pharmingen, San Diego, CA, USA), von Willebrand factor (vWF) (BD Pharmingen), kinase-insert domain receptor (KDR) (R&D Systems, Minneapolis, MN, USA), and CD14 (BD Pharmingen). To identify the cells that expressed these surface antigens, the EPCs were incubated for 40 min at 4 °C in a volume of 100 μl of solution containing an appropriate amount of PE-labeled antibody or corresponding IgG isotype control. At least 1 × 10⁵ EPCs were acquired by using a flow cytometer (Beckman-Coulter, Fullerton, CA, USA).