Anti lin fitc

Umbilical cord blood was collected in several hospitals using Stemcare/CB collect blood bag system (Fresenius Kabi Norge AS) containing citrate phosphate dextrose (CPD) as an anticoagulant. Approval for collection was obtained from the Medical Ethical Committee of the Erasmus University Medical Centre (MEC-2009–410) and written informed consent from the mother was obtained prior to donation of the cord blood. Within 48 hours after collection, mononuclear cells were isolated using ficoll (Lymphoprep, Fresenius Kabi Norge AS). CD34⁺ cells were isolated with double positive immunomagnetic selection using Magnetic Activated Cell Sorting (MACS) technology according instructions of the manufacturer (Miltenyi Biotech GmBH, Bergisch Gladbach, Germany). MACS-selected CD34⁺ cells were either used directly in experiments or stained with anti-Lin-FITC, anti-CD38-PerCP-Cy5.5, anti-CD90-PE (all from eBioscience, Vienna, Austria), anti-CD34-PE-Cy7, anti-CD45RA-APC-H7 (both from BD Biosciences, San Jose, CA, USA) and DAPI (Sigma-Aldrich, St Louis, MO, USA) after which viable DAPI^-Lin^-CD34⁺CD38^lowCD45RA^lowCD90⁺-cells, highly enriched for hematopoietic stem cells (HSC)[30 (link)], were sorted using BD FACSAria Cell Sorting System (BD Biosciences, San Jose, CA, USA).