Generation of Murine and Human Megakaryocytes

Marrow cell generation: marrow was flushed from the bone marrow cavity using PBS and cells were filtered via a 40 μm cell strainer to remove spicules and clumps. After centrifugation, erythrocytes were lysed and then cells were resuspended in complete RPMI medium supplemented with 1% supernatant from the TPO-producing fibroblast cell line GP122 (hereafter called TPO medium) (Villeval et al., 1997 (link)). Murine MK generation: Bone marrow cells were cultured in TPO medium for 4–5 days. MKs were separated from marrow cells using a two-step albumin gradient as described (Schulze, 2016 (link); Shivdasani and Schulze, 2005 (link)). Fetal liver-derived MKs were generated as described (Machlus et al., 2017 (link)). Human MK generation: mobilized peripheral blood or BM CD34 +stem/progenitor cells were purchased from AllCells. 1 × 10⁵ cells were cultured in StemSpan medium supplemented with a MK expansion supplement, both from StemCell for 12–14 days, as described (Liu et al., 2011 (link)). Human neutrophils were obtained from blood from healthy donors. Neutrophil were enriched using a dextran density gradient sedimentation as described (Cunin et al., 2016 (link)). Contaminating red blood cells were lysed by hypo-osmotic shock.