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Immunology and Infection

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

Published: June 23rd, 2013

DOI:

10.3791/50323

1Department of Animal Science, Texas A&M University, 2Department of Biology, Texas A&M University, 3Department of Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Distinct from classical inflammatory responses, macrophages that infiltrate tissues often display polarized activation status that plays a crucial role in regulating host tissue physiological functions1-8. Upon stimulation, macrophage activation can be sorted into classic (M1) and alternative (M2) activation2, 4, 9 . M1 macrophage activation depends on Toll-like receptors (TLRs) and activation of nuclear factor kappa B (NFκB)/c-Jun N-terminal kinase 1(JNK1), leading to production of inflammatory cytokines, such as TNF-α and IL-1β and activation of iNOS that results in increased production of reactive oxygen species such ....

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1. Isolation of Bone Marrow Cells

  1. Isolate femur and tibia bones from 6-8 week old mice, rinse off hair and then cut open the bone.
  2. Use a 21G needle and 10 ml syringe to flush out marrow into cold PBS+2% heat inactivated Fetal Bovine Serum (FBS) (3-5 ml/mouse).
  3. Pass marrow through a 21G needle 4-6 times to dissociate the cells.
  4. Pass cells through a 70 μm cell strainer to remove cell clumps, bone, hair and other cells/tissues.
  5. Add 3 volume of NH4Cl solution .......

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A schematic description of the BMDM generation procedure is presented (Figure 1). High purity of mature macrophages can be observed on day 7 when they represent 95 to 99% of CD11b+F4/80+ cells (Figure 2). Polarized macrophages can be examined using antibodies against CD11b, F4/80, CD11c and CD206 followed by flow cytometry analysis. As shown in Figure 3, M1 macrophages are detected as CD11b+F4/80+CD11c+CD206- cells (Q2), whereas M2 macrophages are CD11b+F4/80+CD11c-.......

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We report here a simple and readily adaptable in vitro procedure to induce activation of macrophages derived from bone marrow progenitor cells. This procedure can be used for investigation of mechanisms responsible for polarization of macrophages. The purity of mature macrophages obtained using this protocol averages 95 to 99%, and no additional purification procedures are required. To investigate the function of specific genes of interests in the context of macrophage polarization, ectopic expression or g.......

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This work was supported by the American Heart Association (BGIA 7850037 to Dr. Beiyan Zhou).

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Name Company Catalog Number Comments
Name of Reagent/Material Company Catalog Number Comments
IMDM Thermo Scientific SH30259.01
Fetal bovine serum Invitrogen 10438-026
Murine GM-CSF PeproTech 315-03
NH4Cl StemCell Technologies 7850
L-929 ATCC CCL-1
70 μm cell strainer BD Biosciences 352350
10 x PBS Thermo Scientific AP-9009-10
Anti-mouse CD11b-APC eBioscience 17-0112-81
Anti-mouse F4/80-FITC eBioscience 11-4801-81
Anti-mouse CD69-PE eBioscience 12-0691-81
Anti-mouse CD86-PE eBioscience 12-0862-81
Propidium Iodine Invitrogen P3566

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