Anti cd73 fitc

UC−MSCs in MCB and WCB were recovered and seeded in T75 flasks at a cell density of 2 × 10⁴ cells/cm². When they reached 90–100% confluence, UC−MSCs were harvested for cell surface marker detection. For UC−MSC sheets, the sheet was digested using TrypLE into single-cell suspension for detection. UC−MSCs were aliquoted into 1 × 10⁶ cells/tube in the staining buffer, consisting of phosphate buffer saline (PBS, Corning (Corning, NY, USA)), supplemented with 1% FBS (Gibco). Then, anti-CD73-FITC (BD (Becton, NJ, USA)), anti-CD90-FITC (BD), anti-CD105-APC (BioLegend (San Diego, CA, USA)), anti-CD11b-FITC (BD), anti-CD19-FITC (BioLegend), anti-CD34-PE (BioLegend), anti-CD45-FITC (BD), anti-HLA-DR-FITC (BD), anti-IgG-FITC (BD), anti-IgG-PE (BD), and anti-IgG-APC (BD) were added to the tubes separately. After staining for 30 min at room temperature in the dark, the cells were washed twice with PBS and resuspended in the staining buffer for flow cytometry analysis.

Flow cytometric analysis was used to assess the expressions of the surface markers on the hBMSCs using an Apogee A50-MICRO flow cytometer (Apogee, UK), as described below. In brief, cells were digested using trypsin, centrifuged, and resuspended in cold phosphate-buffered saline (PBS) containing 1% FBS. After the concentration was adjusted to 1 × 10⁶ cells/mL, the cell suspension was incubated with the following antibodies: 20 μL of anti-CD34-PE, 20 μL of anti-CD45-PE, 5 μL of anti-CD73-FITC, and 2 μL of CD90-FITC (BD Biosciences, USA), respectively, for 30 min in dark at 37 °C. After the cells were washed three times with cold PBS, 100 μL of the single-cell suspension was used for the flow cytometric analysis. Untreated cells were used as a negative control.

To confirm their identities as MSCs, both SHED and OOMDSCs were characterized by flow cytometry for the expression of typical surface markers of MSCs (CD29, CD44, CD90, CD73, CD105, and CD106) and endothelial (CD31) and hematopoietic (C34) cells. All cells were incubated with antibodies (conjugated with fluorochromes) that had the ability to bind specifically to intracellular and cell surface proteins to compare and characterize the cells according to the expression of specific antigens. A total of 1 × 10⁶ cells obtained from cell cultures and diluted in 100 μL of 1XPBS⁻ were transferred to flow cytometry tubes and incubated with the following monoclonal antibodies for 15 minutes at room temperature in the dark for staining: anti-CD29-PE, anti-CD44-PE, anti-CD73-FITC, anti-CD90-FITC, anti-CD105-PE, anti-CD166-PE, anti-CD34-FITC, and anti-CD31-FITC (BD Bioscience, Becton Dickinson Franklin Lakes, NJ). The samples were then washed with 1XPBS⁻, resuspended in 500 μL of 1XPBS⁻, run on a FACSCalibur (BD, Becton Dickinson, Franklin Lakes, NJ) flow cytometer, and subsequently analyzed using FlowJo software (TreeStar Inc.). A sample of unstained cells was prepared for each experiment to eliminate the influence of any nonspecific staining and innate autofluorescence of the cells. As a negative control for the reactions, an isotype control was used for each antibody.