Ab239246

Cells (2 × 10⁵) were incubated with a specific monoclonal antibody conjugated with either fluorescein isothiocyanate (FITC) or phycoerythrin (PE) in 200 μL PBS (SH30256.01; HyClone) for 30 min in the dark at 4°C. The cell surface antigens were then analyzed by flow cytometry. Antibodies against CD34 (PE) (ab187284; Abcam), CD45 (PE) (ab134202; Abcam), CD73 (FITC) (ab239246; Abcam), CD90 (FITC) (ab124527; Abcam), and CD146 (FITC) (ab78451; Abcam) were used. Mouse monoclonal IgG1 (ab170190; Abcam) isotype was used as control.

The hBMSCs were isolated from the bone marrows harvested in the pelvis of the healthy donors (15–85 years old) who underwent osteotomy for health reasons in Linyi People’s Hospital. In brief, under aseptic conditions, 10 mL of the bone marrow was extracted using a 20-mL syringe (containing 2000 IU heparin) and immediately mixed with heparin. The bone marrow was centrifuged at 1200 g for 10 min for the separation of adipose tissues. The bone marrow was then resuspended in 15 mL of DMEM and added into the centrifuge tube with the same volume of Ficoll-Paque™ Plus lymphocyte separation solution (at the density 1.077 g/mL), followed by centrifugation at 2000 g for 20 min. The supernatant containing nucleated cells was collected using a pipette and subsequently washed with phosphate buffer saline (PBS), followed by centrifugation at 1000 g for 8 min. Next, 10 μL of cell suspension was added into 490 μL of PBS. The cells were then seeded in culture flasks at a density of 1 × 10⁵ cells/flask and cultured in a 5-mL low-glucose medium at 37 °C in 5% CO₂ and saturated humidity. The relevant markers for hBMSCs (Abcam Inc., Cambridge, UK) CD90 (ab225), CD105 (ab227388), CD44 (ab25024), and CD73 (ab239246) as well as hemopoiesis markers (Abcam Inc., Cambridge, UK) CD19 (ab245235), CD34 (ab18224), CD45 (an27287), and HLA-DR (ab1182) were used in this study.