Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages
Summary
This protocol describes a non-enzymatic and straightforward method for isolating 7-9-day-old neonatal mouse bone marrow cells and generating differentiated macrophages using a supernatant of L929 cells as a source of granulocyte colony-stimulating factor (M-CSF). The bone marrow-derived macrophages were further analyzed for surface antigens F4/80, CD206, CD11b, and functional competency.
Abstract
Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging and time-consuming, yet for some models, it is translationally relevant and necessary. This protocol describes an efficient and straightforward method for preparing bone marrow cells from 7-9-day-old pups. These cells can then be further isolated or differentiated into specific cell types of interest. Macrophages are crucial immune cells that play a major role in inflammation and infection. During development, neonatal macrophages contribute significantly to tissue remodeling. Moreover, the phenotype and functions of neonatal macrophages differ from those of their adult counterparts. This protocol also outlines the differentiation of neonatal macrophages from the isolated bone marrow cells in the presence of L929-conditioned medium. Surface markers for differentiated neonatal macrophages were assessed using flow cytometric analysis. To demonstrate functionality, the phagocytic efficiency was also tested using pH-sensitive dye-conjugated Escherichia coli.
Introduction
Bone marrow encloses both hematopoietic and mesenchymal stem cell populations that are self-renewable and can be differentiated into various cell lineages. Hematopoietic stem cells in the bone marrow give rise to myeloid and lymphoid lineages1. Mesenchymal stem cells produce osteoblasts (bone), adipocytes (fat), or chondrocytes (cartilage)2. These cells have multiple applications in the field of cell biology and tissue engineering, including gene therapy3,4. Progenitor cells present in the bone marrow differentiate into specific cell types in the presence of lineage-specific growth factors. Erythropoietin promotes the proliferation of erythroid progenitor cells, granulocyte colony-stimulating factor (G-CSF) stimulates the growth of neutrophil colonies, and thrombopoietin regulates the production of platelets as a few examples of lineage-specific growth factors5. Cell surface antigen labeled FACS and magnetic-activated cell sorting (MACS) are well-established methods for isolation and purification of the specific bone marrow-derived cell types6.
Though neonatal studies are advancing toward finding the causes of neonatal deaths and addressing the complications during premature births, direct therapeutic development remains an unmet medical need. Smith and Davis stated, "Pediatric patients remain therapeutic orphans"7. There are several challenges, such as small samples, lifelong effects of the outcome, and ethical issues in obtaining consent in clinical studies of neonates8. Hence, there is a high demand for in vivo and in vitro study models specific to neonates to achieve translational relevance. Because of the similarities between anatomical and tissue levels, short gestational periods, and litter sizes, rodents are the most studied mammalian model system.
Here, we describe a detailed, highly feasible, and reproducible procedure for isolating bone marrow from 7-9-day-old mouse pups and their ability to differentiate into macrophages. However, a variety of cell lineages could be achieved with the use of distinct differentiation signals. We also demonstrate the presence of cell surface markers and the presence of in vitro phagocytic activity expected for bone marrow-derived macrophages (BMDMs).
Protocol
Representative Results
Discussion
Research involving neonatal mouse models can present a number of challenges. Neonates have a developing immune system that is unique compared to adults8. As such, data generated from adult animal models should not be assumed to apply to newborns, and several published works have articulated this idea well18,19. Therefore, neonatal-specific models and sources of cells are necessary to study the intricacies of the early-life immune response….
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This work was supported by the National Institutes of Health [R01 AI163333] to CMR. We acknowledge additional funding support provided to the West Virginia University Flow Cytometry and Single Cell Core Facility by the following grants: WV CTSI grant GM104942, Tumor Microenvironment CoBRE grant GM121322 and NIH grant OD016165.
Materials
| 40 µm strainer | Greiner | 542040 | Cell culture |
| 96 well round (U) bottom plate | Thermo Scientific | 12-565-65 | Cell culture |
| Anti-mouse CD11b-BV786 | BD Biosciences | 740861 | FACS analysis |
| Anti-mouse CD206-Alexa Fluor488 | BD Biosciences | 141709 | FACS analysis |
| Anti-mouse F4/80-PE | BD Biosciences | 565410 | FACS analysis |
| Countess3 | Thermo Scientific | TSI-C3ACC | Automated cell counter |
| DMEM | Hyclone | SH30022.01 | Cell culture |
| DMSO | VWR | WN182 | Cell culture |
| DPBS, 1x | Corning | 21-031-CV | Cell culture |
| Escherichia coli O1:K1:H7 | ATCC | 11775 | Infection |
| EVOS FL | Invitrogen | 12-563-649 | Cell Imaging System |
| FBS | Avantor | 76419-584 | Cell culture |
| FluoroBright BMDM | Thermo fisher Scientific | A1896701 | Dye free culture media |
| Glutamine | Cytiva | SH30034.01 | Cell culture |
| HEPES | Cytiva | SH30237.01 | Cell culture |
| L-929 | ATCC | Differentiation | |
| LSRFortessa | Becton Dickinson | Flowcytometer | |
| Lysotracker red DND 99 | Invitrogen | L7528 | Fluorescent dye |
| MEM | Corning | 15-010-CV | Cell culture |
| Penicillin /streptomycin | Hyclone | SV30010 | Cell culture |
| pHrodo green STP ester | Invitrogen | P35369 | Fluorescent dye |
| T75 flask | Cell star | 658170 | Cell culture |
| Trypsin-EDTA | Gibco | 25300120 | Cell culture |
| Zeiss 710 | Zeiss | P20GM103434 | Confocal |
Referencias
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