Mac ls column

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)}

Murine bones (2× femura and 2x tibiae) were removed and bone marrow was flushed in KDS-BSS with 2% FCS. 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) in KDS-BSS 2% FCS, followed by anti-biotin magnetic microbeads (Miltenyi Biotec, NSW, Australia) and MAC LS columns (Miltenyi Biotec). Single cell suspensions were cultured for 3–5 days at 5 × 10⁵ cells per ml in serum-free medium (StemPro^TM-34 SFM), supplemented with 100 ng/ml murine TPO (Peprotech) at 37 °C, 5% CO₂ and mature megakaryocytes purified using a discontinuous BSA density gradient (3, 1.5 and 0%). Cells were harvested in the 3% layer after 35 min.

CD11c⁺ and T cells were purified by magnetic cell sorting. To purify CD11c⁺ cells, BM culture cells were incubated with anti-CD11c (N418) MAC beads (10 µl/10⁷ cells) (Miltenyi Biotec, Auburn, CA) in the presence of 2% FCS and 2 mM EDTA in PBS at 4°C for 15 min. Cells were positively selected with the MAC LS column and the magnetic separator (Miltenyi Biotech). CD11c⁺ cell purity was at least 93%. T cells were isolated from spleen. Briefly, splenocytes were incubated with a cocktail of biotin-conjugated antibodies and anti-biotin microbeads (Miltenyi Biotech) to label non-T cells. Cells were negatively selected with the LS column as described. T cell purity was >90%.