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

Part of the Methods in Molecular Biology book series (MIMB,volume 1989)


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.

Key words

  • Bone marrow
  • Hematopoiesis
  • High-dimensional cytometry
  • Mass cytometry
  • CyTOF
  • Cell cycle

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We wish to thank Dr. Paula Niewold for critical reading of this manuscript, and assistance with many of the experiments that lead to the development of this protocol, and to Rebecca Burrell for assistance with experiments. We would like to thank Dr. Philip Hansbro, Dr. Nicole Hansbro, Dr. Markus Hofer, David Jung, Tamara Suprunenko, Dr. Iain Campbell, and Vickie Xie for provision of bone marrow samples from various disease models over the years. We would like thank Dr. Fabian Held for assistance in troubleshooting our tSNEplots script, as the use of such a tool expedited our analysis of bone marrow data. We would also like to express gratitude to Dr. Sebastian Stifter, Dr. Carl Feng, Dr. Ben Roediger, Dr. Sean Bendall, Dr. Greg Behbehani, Dr. Helen Goodridge, and Dr. Alberto Yáñez for discussions on the nature of bone marrow hematopoiesis over the years, as these discussions have been invaluable. We would like to thank the Sydney Cytometry Facility, Ramaciotti Facility for Human System Biology, and Caryn van Vreden for assistance with mass cytometry and provision of reagents.

Thomas Ashhurst was supported by an Australian Postgraduate Award (APA) during the early development of parts of this protocol, and is currently supported by the International Society for the Advancement of Cytometry (ISAC) Marylou Ingram Scholars program. Darren Cox was also supported by an APA. This work was funded by National Health and Medical Research Council (NHMRC) funding.

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Correspondence to Nicholas J. C. King .

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Ashhurst, T.M., Cox, D.A., Smith, A.L., King, N.J.C. (2019). Analysis of the Murine Bone Marrow Hematopoietic System Using Mass and Flow Cytometry. In: McGuire, H., Ashhurst, T. (eds) Mass Cytometry. Methods in Molecular Biology, vol 1989. Humana, New York, NY.

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