Macs immunomagnetic absorption column separation device

The PQE60 plasmid and E.Coli BL 21 were obtained from QIAGEN (Valencia, CA, USA). Isopropyl-1-thio-ß- D-galactopyranoside (IPTG) was from Bio-Basic Inc (Amherst, NY, USA) to induce recombinant SCF expression in bacteria. Guanidine Hydrochloride was from Amresco Inc (Solon, OH, USA). Urea was from Shanghai Experimental Reagent Inc (Shanghai, China). RPMI 1640 medium and STEM PRO®-34 SFM medium were from Gibco (Grand Island, NY, USA). Fetal bovine serum was from Hyclone (Logan, UT, USA). Goat-anti-mouse immunoglobulin G (IgG) conjugated with alkaline phosphatase was from Biolegend (Canada). Prestained protein molecular weight marker was from Bio-Rad (USA). Biotin conjugated goat anti-mouse-IgG was from Biolegend (San Diego, CA, USA). Mouse monoclonal antibody isotyping reagent kit and streptavidin-peroxidase was from Sigma-Aldrich (St. Louis, MO, USA). MACS immunomagnetic absorption column separation device and CD34 MicroBead Kit, was from Miltenyi Biotec (Germany). rhTPO and rhFLT-3 were from Pepro Tech (USA). Anti-human CD34 mAb conjugated with phycoerythrin was from Immunotec (Canada). Peptides were synthesized by Nanjing Genscript Inc. (Nanjing, China).

Fresh UCB samples were obtained within 6–8 hours of delivery from Suzhou Municipal Hospital (Suzhou, China). UCB CD34⁺ cells were isolated from total mononucleated cells (MNC) with the MACS immunomagnetic absorption column separation device and CD34 MicroBead Kit, according to the manufacturer’s instructions (Miltenyi Biotec, Germany). MNCs were obtained by density centrifugation, with use of Ficoll-Hypaque Premium reagent (GE healthcare, USA). The purity of CD34⁺ cells was verified using flow cytometry, with an anti-human CD34 mAb conjugated with phycoerythrin (PE, Immunotec, Canada) and a model BD FACSVerse flow cytometer (BD, USA).