Pe mouse anti human cd309

ECFCs scraped by Corning cell lifter (Sigma, St. Louis, MO, USA) were suspended in PBS and 1 × 10⁶ cells were stained by specific antibodies at 4 °C for 20 minutes according to the manufacturer’s instructions. The antibodies used were FITC Mouse anti-Human CD34 (BD Pharmingen, Franklin Lakes, NJ, USA), FITC Mouse anti-Human CD31 (BD Pharmingen), FITC Mouse anti-Human CD45 (BD Pharmingen), FITC Mouse anti-Human CD144 (BD Pharmingen), PE Mouse anti-Human CD309 (BD Pharmingen), FITC Mouse IgG1, κ Isotype Control (BD Pharmingen), PE Mouse IgG1, κ Isotype Control (BD Pharmingen). To obtain single cell suspensions for FACS, cells were passed through a 40-μm cell strainer and transferred to a 5 ml polystyrene round-bottom Falcon tube (BD Pharmingen). ECFC surface antigens were detected by BD FACSCalibur (BD Biosciences, San Jose, CA) and the percentages of cell expressing the surface markers were calculated by FlowJo (BD Biosciences).

For characterization of isolated cells, the surface markers of EPCs were analyzed by using flow cytometric assay. After day-7 of culture bone marrow cells in EPC-associated medium, cells were detached with 0.25% Trypsin-EDTA solution, neutralized by FBS, and washed twice with PBS. Thereafter, a panel of antibodies directed against PE mouse anti-human CD309 (BD Pharmingen), PE mouse anti-human CD31 (ebioscience), FITC mouse anti-human CD133 (Miltenyi BioTec) and Tie-2 (mouse anti-human Tie-2, Abcam) and goat anti-mouse IgG-Texas red (Abcam) were provided. Briefly, an approximate number of 5 × 10⁵ to 10⁶ cells blocked by 2% FBS solution for 15 minutes, and incubated with 100 µL PBS containing manufacturer’s recommended concentration for 30 minutes, washed twice with PBS and fixed in 4% paraformaldehyde (PFA). Ultimately, the percentage of EPC marker positive cells analyzed by BD FACSCalibur^™ flow cytometer and final raw data processed via FlowJo software version 7.6.1.