Mouse anti human cd73

Flow cytometry was implemented for measuring UC-MSC surface markers. Briefly, cells were incubated with FITC-conjugated primary antibodies after preprocessing steps as described in the section of cell cycle analysis. Then, the cells were subjected to a MACSQuant Analyzer Flow Cytometer (Miltenyi Biotec) for analysis. The primary antibodies used in this experiment are mouse anti-human CD34 (555821; BD Pharmingen), mouse anti-human CD45 (555482; BD Pharmingen), mouse anti-human CD73 (561254; BD Pharmingen), mouse anti-human CD90 (561969; BD Pharmingen), mouse anti-human CD105 (561443; BD Pharmingen), and mouse anti-human HLA-DR (555560; BD Pharmingen).

Primary culture of the USCs was performed as previously described [33 (link),34 (link)]. Briefly, the USCs were obtained from four young male adult donors (20–30 years old). Fresh urine sample (about 200 mL) was centrifuged and washed with PBS for twice. The sediment was re-suspended and cultured in T25 cell culture flasks with 5 mL of established medium [35 (link)].
Cells in logarithmic growth phase were digested with trypsin (Gibco, USA), with their density adjusted to 1 × 10⁶ cells/mL with PBS. 100 μL aliquot of cell suspension was moved to each flow tube (Corning, USA). Thereafter, 2 μL of mouse anti-human CD29 (559883, BD Pharmingen™, USA), mouse anti-human CD34 (555821, BD Pharmingen™), mouse anti-human CD44 (555478, BD Pharmingen™), mouse anti-human CD73 (550257, BD Pharmingen™), mouse anti-human CD90 (561558, BD Pharmingen™) and mouse anti-human CD133(130-111-085, Miltenyi Biotec GmbH, Germany) antibodies were added to each tube. The mixture was allowed to incubate for 30 min in darkness at room temperature. Thereafter, the cells were washed twice with PBS and re-suspended in 200 μL PBS for flow analysis.