Foetal calf serum

Foetal livers were removed at embryonic day (E) 13.5 and transferred into Dulbecco's modified Eagle's medium (high glucose version) with 10% foetal calf serum (Gibco, Paisley, UK). Bone marrow was flushed into Dulbecco's modified Eagle's medium with 2% foetal calf serum. The cells were lineage-depleted by incubation with a mix of biotinylated antibodies (CD4, CD2, CD3, CD5, CD8, Ter119, B220, CD19, Gr-1, Ly6G, F4/F8, CD127; WEHI mAb Facility) in KDS-BSS 2% foetal calf serum, followed by anti-biotin magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and MAC LS columns (Miltenyi Biotec) in EDTA-KDS-BSS 0.5% foetal calf serum. Single cell suspensions were cultured for 3–5 days at 5 × 10⁵ cells per ml in serum-free medium^{47 (link)} supplemented with 100 ng/ml murine thrombopoietin (WEHI) at 37 °C, 5% CO₂, and mature megakaryocytes purified using a BSA gradient as described.^{5 (link)}