Cd45 pe

The cells were cultured in high glucose α-MEM (Gibco, USA), 10% FBS (Gibco, USA), 100 U/ml penicillin and 0.1 mg/ml streptomycin for 1 week, culture medium was replaced twice a week. Cells were collected and counted, then re-suspended with α-MEM, we adjusted the cell density at 1 × 10⁶ cells/ml. Each 0.1 ml sample was incubated with 20 μl of CD73-PE, CD90-FITC, CD34-PE, CD45-PE, CD105-PE (Santa Cruz, USA) at 4 °C for 1 h. Antibody binding was analyzed by flow cytometry (Becton Dickson, USA).

To analyze the cell surface antigen expressions, the cells from the second passage were harvested by trypsin/EDTA treatment for 4 min at 37°C then washed twice with PBS. 1 × 10⁵ cells/tube were incubated with the conjugated antibody for 20 min on ice in the dark. The cell suspensions were washed twice, resuspended in 2% FBS/PBS, and analyzed using a flow cytometry cell sorting Vantage cell sorter (BD Biosciences, San Jose, CA). Data were analyzed using the FACS software (FlowJo LLC, Ashland, OR). The following conjugated antibodies were used: STRO-1-PE (Santa Cruz Biotech, sc-47733, Santa Cruz, CA), CD-29-PE (BD Biosciences, Cat. No. 557332, San Jose, CA), CD-90-PE (BD Biosciences, Cat. No. 555596), CD105-FITC (BD Biosciences, Cat. No. 561443), CD-34-PE (BD Biosciences, Cat. No. 560941), CD-45-PE (Santa Cruz Biotech, sc-28369), FITC-conjugated IgG control antibody (BD Biosciences, Cat. No. 555748), and PE-conjugated IgG control antibody (BD Biosciences, Cat. No. 555749). Isotype control antibodies were used as negative controls.