Pe anti human cd133

The phenotype of primitive hematopoietic cells was evaluated at different time points during the ex vivo expansion based on the cell membrane expression of specific antigens. The cells were harvested from the culture, centrifuged (HERAEUS Multifuge X3R, Thermo Scientific, Illkirch, France) at 300× g for 5 min, and labeled 15 min at room temperature (RT) with BV480 anti-human CD45RA (BD Biosciences, Le Pont de Claix, France), APC-Alexa Fluor 750 anti-human CD34 (Beckman Coulter, Villepinte, France), PE anti-human CD133 (BD Biosciences), and 7AAD (Exbio-Clinisciences, Nanterre, France). The cells were washed with PBS, centrifuged for 5 min at 300× g and analyzed by flow cytometry with a FACS Canto II (BD Biosciences). The FACS data were processed with FlowJo^TM software version 10.8.0 (BD Biosciences).

The pretreated cells were harvested and washed twice with FCM buffer (PBS with 5% FBS and 0.1% NaN3). Cells were resuspended in PBS and incubated with PE-anti-human CD133 (BD Biosciences) or isotype control antibodies for 30 min at 4°C. CD133+ cells were sorted with a MoFlo XDP cell sorter (Beckman Coulter) and cultured in serum-free medium supplemented with 20 ng/mL human recombinant EGF and 10 ng/mL human recombinant bFGF.