Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

Summary

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.

Abstract

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.

Introduction

Distinct from classical inflammatory responses, macrophages that infiltrate tissues often display polarized activation status that plays a crucial role in regulating host tissue physiological functions^1-8. Upon stimulation, macrophage activation can be sorted into classic (M1) and alternative (M2) activation^{2, 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 ....

Protocol

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 (0.8% NH₄Cl solution, Stemcell Technology), and incubate on ice for 10 min to remove red blood cells.
6. Spin down cells at 500 x g ....

Results

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-.......

Discussion

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.......

Materials

List of materials used in this article

Name	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