Analysis of the Murine Bone Marrow Hematopoietic System Using Mass and Flow Cytometry.

Abstract

The hematopoietic system produces erythrocytes (red blood cells), leukocytes (white blood cells), and thrombocytes (platelets) throughout the life of an organism. Long-lived hematopoietic stem cells give rise to early progenitors with multi-lineage potential that progressively differentiate into lineage-specific progenitors. Following lineage commitment, these progenitors proliferate and expand, before eventually differentiating into their mature forms. This process drives the up- and downregulation of a wide variety of surface and intracellular markers throughout differentiation, making cytometric analysis of this interconnected system challenging. Moreover, during inflammation, the hematopoietic system can be mobilized to re-prioritize the production of various lineages, in order to match increased demand, often at the expense of other lineages. As such, the response of the hematopoietic system in the bone marrow (BM) is a critical component of both immunity and disease. Because of the complexity of the hematopoietic system in steady state and disease, high-dimensional cytometry technologies are well suited to the exploration of these complex systems. Here we describe a protocol for the extraction of murine bone marrow, and preparation for examination using high-dimensional flow or mass cytometry. Additionally, we describe methods for performing cell cycle assays using bromodeoxyuridine (BrdU) or iododeoxyuridine (IdU). Finally, we describe an analytical method that allows for a system-level analysis of the hematopoietic system in steady state or inflammatory scenarios.