Bovine serum albumin insulin transferrin

Leukapheresis products were obtained from CML patients at the time of diagnosis and bone marrow from healthy donors was collected as a normal control. All samples were obtained with ethical approval from the Research Ethics Committee, Northern Ireland, and the National Health Service Greater Glasgow Institutional Review Board and those involved gave their informed consent in accordance with the Declaration of Helsinki. CD34⁺ cells were enriched from the samples using an AutoMACS system according to the manufacturer's instructions (Miltenyi Biotech, Surrey, UK). CD34⁺ cells were stained with CD34-APC and CD38⁻ FITC (BD Biosciences, Oxford, UK) and sorted using a FACSAria (BD Biosciences) to obtain an enriched CD34⁺38⁻ population. CD34⁺ and CD34⁺38⁻ cells were cultured in IMDM medium (Gibco BRL) supplemented with serum substitute (bovine serum albumin, insulin, transferrin; StemCell Technologies, Grenoble, France), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.1 mM 2-mercaptoethanol and 0.8 μg/ml low-density lipoprotein (all from Sigma-Aldrich, Dorset, UK) and a growth factor cocktail of 20 ng/ml recombinant human (rh) IL-3, 20 ng/ml rhIL-6, 100 ng/ml rh Flt-3 ligand and 100 ng/ml rh stem cell factor (all from StemCell Technologies).