Recombinant human interleukin 3

Patient samples were obtained from the Manchester Cancer Research Centre Biobank (HTA 30004) and had ethical approval from the National Research Ethics Service committee (14/LO/0489). Experimental details are available in Supplementary Methods. CD34⁺ cells were enriched using CliniMACS (Miltenyi Biotec, Bisley, UK) and colony-forming assays performed in methylcellulose complete media (R&D Systems, Abingdon, UK) supplemented with 2 U/ml erythropoietin at a density of 3000 cells per ml. To assess retention of self-renewal capacity, the resulting colonies at day 7 were replated in methylcellulose and colonies counted at 14 days. CD34⁺ cells were stained using CellTrace Violet Cell Proliferation Kit (Molecular Probes, Thermo Fisher) and then seeded at a density of 1.5 × 10⁵/ml in Iscove’s modified Dulbecco’s medium, 20% (v/v) fetal calf serum, recombinant human interleukin-3 (20 ng/ml), recombinant human stem cell factor (50 ng/ml) and Flt-3 (Fms-related tyrosine kinase 3) ligand (10 ng/ml) (PeproTech, London, UK). On day 0 and following 8 days of culture, cells were harvested and stained with CD34-APC (eBioscience, Thermo Fisher) and appropriate controls using standard protocols. Fluorescence was measured on a LSRFortessa (Becton Dickenson, Oxford, UK) flow cytometer. Results were analysed using FloJo software (Ashland, OR, USA).

Flow sorted primary mast cells were maintained in human mast cell culture media (StemPro^TM-34 Serum-Free media (Gibco), StemPro-34 Nutrient Supplement (Gibco), L-Glutamine (2 mM) (Gibco), Penicillin (100 U/mL)/Streptomycin (100 µg/mL) (Gibco), recombinant human stem cell factor (100 ng/mL) (PeproTech), recombinant human Interleukin 6 (100 ng/mL) (PeproTech) and Recombinant human Interleukin-3 (30 ng/mL) (PeproTech)) at 37 °C in 5% CO₂, with media changes every 5–7 days [22 (link)]. Primary mast cell numbers were determined with TC-20^TM Automated Cell Counter system (Bio-Rad) according to manufacturer’s protocol at every media change. Growth curves for primary mast cells are shown in Figure S3a.