Isolation, In Vitro Differentiation, and Culture of Murine Megakaryocytes From Fetal Liver and Adult Bone Marrow

Abstract

AbstractMegakaryocytes (MKs) are the source of circulating platelets and are readily recognized by their large size and distinctive morphology. Their poor representation in hematopoietic tissues often requires enrichment or considerable ex vivo expansion to generate cells for biochemical and cell biological studies. These experimental protocols describe the enrichment of primary MKs directly from the murine bone marrow as well as in vitro differentiation of fetal liver– or bone marrow–derived hematopoietic stem cells into MKs. Although in vitro–differentiated MKs are not synchronized in their maturation, they can be enriched over an albumin density gradient, and one‐third to one‐half of recovered cells will typically elaborate proplatelets. Support protocols describe methods for preparing fetal liver cells, identifying mature rodent MKs by staining for flow cytometry analysis, and immunofluorescence staining of fixed MKs for confocal laser scanning microscopy. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Isolation of mature bone marrow megakaryocytes by magnetic‐activated cell sortingBasic Protocol 2: Preparation of a megakaryocyte suspension culture from murine fetal livers or lineage‐depleted adult bone marrowSupport Protocol 1: Preparation of a single‐cell suspension from murine fetal livers for megakaryocyte cultureSupport Protocol 2: Megakaryocyte culture from lineage‐depleted murine bone marrowSupport Protocol 3: Quality control of megakaryocyte culture with flow cytometrySupport Protocol 4: Immunofluorescence staining of megakaryocytes for detection with confocal laser scanning microscopy