Cd34 pe

The purity of the Dynabead-selected HSCs and differentiated erythroid cells was determined using cell-surface antibodies and flow cytometry (FACSCalibur, BD Biosciences, USA). Briefly, 5 × 10⁴ HSCs were stained with phycoerythrin-conjugated CD34 (CD34-PE) and fluorescein isothiocyanate-conjugated CD45 (CD45-FITC) (BD Biosciences, USA) according to the manufacturer’s instructions and analysed. Subsequent to erythroid differentiation, markers of the derived-erythroid cells were analysed on day 15 using CD34-PE, CD45-APC, CD71-FITC and CD235a-PE (BD Biosciences, USA). The controls included in the flow cytometry experiments were as follows: (i) No cells (cell suspension fluid: 1x PBS), (ii) Cells only, (iii) Cells and primary antibody only, (iv) Cells and secondary antibody only. These were included to ensure specificity in antigen–antibody interactions in the experiments. During optimization, a separate cell line, RAMOS (RA-1 CRL1596, ATCC) was used to independently confirm the specificity of the primary antibodies (CD34, CD45, CD71 and CD235a) compared to K562 cells (using ATCC reference expression levels and those determined in our lab) and HSC-derived erythroid cells.

The phenotype of rDFSCs and rDPCs was identified by flow cytometric analysis. The MSC phenotyping cocktail comprised both positive (CD29-FITC, CD44/CD90-PE, BD Bioscience, USA) and negative (CD34-PE, CD45-FITC, BD Bioscience, USA) fluorochrome-conjugated monoclonal antibodies. IgG1-FITC and IgG1-PE (BD Bioscience, USA) were used as isotype controls. Third-passage rDFSCs and rDPCs were suspended to 5 × 10⁵ cells/mL in PBS solution, stained with different antibodies for 30 min at 4 °C, washed with PBS, resuspended in FACS buffer, and analyzed using a MOFlo™ high-performance cell sorter (Beckman Coulter, USA).

Expression of cell surface markers on the hUC-MSCs was analyzed using flow cytometry. The following monoclonal antibodies (mouse anti-human) were used for flow cytometric immunophenotyping of human mesenchymal stem cells-extracellular vesicles (hMSCs-EV) and human mesenchymal stem cells (hMSCs): CD73 FITC, CD105 PE, CD45FITC, and CD34 PE (BD Bioscience, USA). The cells were characterized with regard to some positive MSC surface markers, including CD105 and CD73, and negative for CD34 and CD45 (hematopoietic markers). hUC-MSCs were suspended in PBS containing 5% bovine serum albumin (Sigma-Aldrich, USA) at a concentration of 3 × 10⁵ cells/50 μL and stained with these markers, respectively. Labeled cells were acquired using a FACS flow cytometer (BD Biosciences, USA) for acquisition and analyzed using FlowJo Software (Tree Star) [26 (link)].