Platelet and Megakaryocyte Isolation and Analysis

Platelets were purified from blood collected by cardiac puncture, and counted by flow cytometry as previously described (Gutiérrez-Herrero et al., 2020 (link)). Bone marrow cells (BMCs) were obtained from the femora and tibiae of mice and MKs were purified by BSA density gradient as described (Gutiérrez-Herrero et al., 2020 (link)). Alternatively, MKs were obtained in vitro by culturing BMCs in RPMI with 10% horse serum, supplemented with 50 ng/ml recombinant TPO, 10 ng/ml IL-3 (Interleukin-3, Invitrogen), 10 ng/ml SCF (Stem Cell Factor), 10 ng/ml IL-11 and 10 ng/ml IL-6 (Miltenyi) for 6 days. MKs were detected by flow cytometry with anti-CD41-FITC (MWReg30) and anti-CD61-PE (2C9.G3) antibodies, or anti-CD42b-FITC antibody, previous gate with anti-CD61-PE antibody. Ploidy analysis was performed as described (Ortiz-Rivero et al., 2018 (link); Gutiérrez-Herrero et al., 2020 (link)).