Characterization of neutrophils and macrophages from ex vivo-cultured murine bone marrow for morphologic maturation and functional responses by imaging flow cytometry.
Neutrophils and macrophages differentiate from common myeloid progenitors in the bone marrow, where they undergo nuclear morphologic changes during maturation. During this process, both cell types acquire critical innate immune functions that include phagocytosis of pathogens, and for neutrophils the release of nuclear material called nuclear extracellular traps (NETs). Primary cells used to study these functions are typically purified from mature mouse tissues, but bone marrow-derived ex vivo cultures provide more abundant numbers of progenitors and functionally mature cells. Routine analyses of these cells use conventional microscopy and flow cytometry, which present limitations; microscopy is laborious and subjective, whereas flow cytometry lacks spatial resolution. Here we describe methods to generate enriched populations of neutrophils or macrophages from cryopreserved mouse bone marrow cultured ex vivo, and to use imaging flow cytometry that combines the resolution of microscopy with flow cytometry to analyze cells for morphologic features, phagocytosis, and NETosis.
Department of Biological Sciences, Biomedical Engineering and Biotechnology Program, University of Massachusetts Lowell, Lowell, MA, USA.
Margery G H
MG
Pelletier
Department of Biological Sciences, Biomedical Engineering and Biotechnology Program, University of Massachusetts Lowell, Lowell, MA, USA.
Klaudia
K
Szymczak
Department of Biological Sciences, Biomedical Engineering and Biotechnology Program, University of Massachusetts Lowell, Lowell, MA, USA.
Anna M
AM
Barbeau
Integrative Physiology Laboratory, Combat Feeding Directorate, U.S. Army Natick Soldier RDEC, Natick, MA, USA.
Gianna N
GN
Prata
Integrative Physiology Laboratory, Combat Feeding Directorate, U.S. Army Natick Soldier RDEC, Natick, MA, USA.
Kevin S
KS
O'Fallon
Department of Biological Sciences, Biomedical Engineering and Biotechnology Program, University of Massachusetts Lowell, Lowell, MA, USA. Electronic address: peter_gaines@uml.edu.
Peter
P
Gaines
D000818:Animals; D001854:Bone Marrow Cells / Q000187:drug effects / Q000276:immunology* / Q000648:ultrastructure; D000001:Calcimycin / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D015925:Cryopreservation; D065206:Extracellular Traps / Q000000981:diagnostic imaging* / Q000276:immunology; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D019044:Image Cytometry / Q000295:instrumentation / Q000379:methods*; D007113:Immunity, Innate; D008264:Macrophages / Q000187:drug effects / Q000276:immunology* / Q000648:ultrastructure; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology* / Q000648:ultrastructure; D010587:Phagocytosis; D061251:Primary Cell Culture; D013194:Staining and Labeling / Q000379:methods; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D005456:Fluorescent Dyes; D000001:Calcimycin; D013755:Tetradecanoylphorbol Acetate
Cell morphology; Fluorescence microscopy; Myeloid; NETosis; Nuclear decondensation; Phagocytosis
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