Peripheral blood mononuclear cells (PBMCs) were isolated through Ficoll-Hypaque (Sigma) density centrifugation from blood obtained from healthy volunteers. CD34+ cells were enriched from PBMCs using positive magnetic affinity column purification (Miltenyi Biotec) and eosinophil progenitors were derived using the technique of Hudson et al. (22 (link)) by culturing purified CD34+ cells in complete medium (RPMI1640 and 10% FBS) supplemented with stem cell factor (SCF; 25 ng/ml; BD Biosciences), thymopoietin (TPO; 25 ng/ml; R&D System, Minneapolis, MN), Fms-like tyrosine kinase 3 (Flt3) ligand (25 ng/ml; BD Biosciences), IL-3 (25 ng/ml; BD Biosciences) and IL-5 (25 ng/ml; BD Biosciences) with or without IFN-γ (20 ng/ml; BD Bioscience) for 3 days and then cultured for an additional 3 weeks with just IL-3 and IL-5 (±IFN-γ). Cells were washed and fresh media and cytokines were applied weekly. Maturation was accessed as previously described (17 (link)) and cells were activated as described above.
Mast Cells. For studies involving mast cells, CD34+ cells isolated as described were cultured for 8 weeks with stem cell factor and IL-6 and, for the first week only, with IL-3 according to the methodology of Kirshenbaum et al (23 (link), 24 (link)).
Mast Cells. For studies involving mast cells, CD34+ cells isolated as described were cultured for 8 weeks with stem cell factor and IL-6 and, for the first week only, with IL-3 according to the methodology of Kirshenbaum et al (23 (link), 24 (link)).