Anti human cd34 antibody

Bone marrow samples were collected from patients newly diagnosed with CML as part of an institutionally approved protocol for cell sample collection (Centre Hospitalier Nice, 06204 Nice, France). Cord blood and bone marrow samples were collected from healthy donors with informed consent. Mononuclear cells were isolated by density centrifugation (Ficoll-Paque Plus, STEMCELL Technologies), washed with PBS with 5% fetal calf serum (FCS). For clonogenic assays CD34⁺ cells were positively enriched with CD34 microbeads (CD34 MicroBead Kit, Miltenyi Biotec). For LTC-IC, CD34⁺ cells were first positively enriched and then depleted for CD38⁺ cells (human CD34⁺CD38⁻ isolation kit, Miltenyi Biotec). The CD34⁺ cells, after sorting, were stained with anti-human-CD34 antibody (Miltenyi Biotec, 130-113-741) and checked by flow cytometry to ensure purity (always above 96%).

CB was collected at the Mater Hospital in Brisbane following full-term births with the informed written consent from mothers. Ethics approval was granted by the Mater Health Services Human Research Ethics Committee and the Queensland University of Technology Human Ethics Committee, with all methods carried out in accordance with the approved guidelines (Ethics number: 1100000210). CD34⁺ cell isolation from CB was performed within 24 hours of collection. CB was diluted with PBS containing 2 mM EDTA and mononucleated cells were isolated by Ficoll-Paque (GE Healthcare) density centrifugation. CD34⁺ progenitor cells were purified using the CD34 MicroBead Kit-UltraPure and AutoMACS Pro Seperator (both from Miltenyi) as per manufacturer’s instructions. CD34⁺ cells were cryopreserved in 10% DMSO and 90% FBS, slowly frozen to −80 °C overnight and stored in liquid nitrogen until use. CD34⁺ cells were pooled from multiple CB donors to obtain sufficient cells for experiments. CD34-purity was assessed prior to cell culture by anti-human CD34 antibody (Miltenyi) staining and flow cytometry on the LSRII (Becton Dickinson), achieving more than 90% purity.