1.Preface
Macrophages are large phagocytes differentiated from monocytes in the blood. They are present in almost all tissues, especially those that are in frequent contact with the outside world such as skin, lungs and intestines. Macrophages play a vital role in many biological processes such as human immune defense, inflammatory response, tissue repair, immune regulation and disease development.
Primary human macrophages are difficult to isolate from tissues in sufficient numbers and do not proliferate in culture. Monocyte-derived macrophages provide an excellent alternative because monocytes in human blood are easily obtained in large quantities and can be differentiated into macrophages in vitro.
2.Biological functions of macrophages
- Phagocytosis
Macrophages recognize and engulf pathogens and dead cell debris through receptors on their surface. This process not only helps clear infection, but also prevents these substances from accumulating in the body, which could cause inflammation or other problems.
- Anti-pathogen defense
Macrophages fight pathogens by producing chemicals that kill microorganisms, such as reactive oxygen and nitrogen oxides, as well as cytokines and chemokines that regulate inflammatory responses.
- Inflammation regulation
Macrophages play a complex role in inflammatory responses. They can not only promote inflammation by releasing pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin 1 (IL-1), but also inhibit inflammation by producing anti-inflammatory cytokines such as IL-10 and TGF-β.
- Tissue repair and reconstruction
After an inflammatory response, macrophages help repair and regenerate tissues by secreting various growth factors. They clear the remnants of damage while promoting the formation of new tissues.
- Immunomodulation
Macrophages promote specific immune responses by presenting antigens to T cells. At the same time, they regulate other components of the immune system by secreting various cytokines, such as regulating the activity of T cells and B cells.
- Relationship with diseases
Macrophages play an important role in the development of many diseases, including infectious diseases, autoimmune diseases, tumors, and chronic inflammatory diseases such as atherosclerosis.
3. Classification of macrophages
According to their activation state and function, macrophages can be divided into two major categories: M1 and M2. These two subtypes play different roles in immune response and inflammation regulation.
M1 macrophages
M1 macrophages are mainly stimulated by cytokines such as interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and have pro-inflammatory characteristics. They have strong antimicrobial activity, can produce oxygen free radicals and inflammatory factors, and participate in killing and clearing pathogenic microorganisms such as bacteria and viruses.
M1 macrophages participate in the body's immune defense and inflammatory response, promote the infiltration of inflammatory and immune cells, assist in clearing infected and damaged tissues, and promote the occurrence and maintenance of immune inflammatory responses.
M2 macrophages
M2 macrophages are mainly stimulated by cytokines such as interleukin-4 (IL-4) and interleukin-13 (IL-13), and have anti-inflammatory and repair characteristics. They participate in tissue repair and regeneration processes, promote anti-inflammatory responses and immune regulation.
M2 macrophages play an important role in the late stage of inflammation and tissue repair, promote the resolution of inflammation and tissue repair, regulate the balance of immune responses, and promote tissue regeneration and repair.
Figure 1. Summary of the main macrophage polarization states of activated macrophages [1]
4. Isolation, culture and differentiation of macrophages in vitro
4.1 Separation method
- Isolation from peripheral blood
Isolation of monocytes: Peripheral blood mononuclear cells (PBMCs) are isolated using density gradient centrifugation (e.g., Ficoll-Paque). The blood sample is added to the upper layer of Ficoll, and after centrifugation, the monocytes are located between the plasma and Ficoll layers.
Isolation of macrophages: After the monocytes are isolated from PBMCs, the mononuclear precursor cells can be further isolated by plastic adhesion. Monocytes are cultured in plastic culture dishes for several hours, non-adherent cells are removed, and the remaining adherent cells are mainly mononuclear precursor cells.
- Isolation from tissues
Tissue samples are broken down into single-cell suspensions by mechanical and/or enzymatic treatment (e.g., collagenase and DNase). Non-target cells are removed by density gradient centrifugation or negative selection (using antibodies and magnetic beads) and macrophages are collected.
4.2 Culture method
Commonly used culture media include RPMI 1640 or IMDM, which usually need to be supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and necessary growth factors. Culture conditions are usually in an incubator at 37°C and 5% CO2
4.3 Differentiation induction method
- Differentiation from mononuclear precursor cells
Using M-CSF: Add macrophage colony stimulating factor (M-CSF) to the culture medium, usually at a concentration of 20-50 ng/mL, and continue culturing for 7-10 days to induce the differentiation of mononuclear precursor cells into macrophages.
Using GM-CSF: Granulocyte-macrophage colony stimulating factor (GM-CSF) can also induce the differentiation of macrophages, especially the tendency to produce M1 (pro-inflammatory) macrophages.
- Further regulation of phenotype and function
M1 macrophages: can be induced by adding IFN-γ (interferon-γ) and LPS (lipopolysaccharide) to the culture medium.
M2 macrophages: can be induced by adding anti-inflammatory cytokines such as IL-4 and IL-13.
These methods allow researchers to study the various biological functions of macrophages and their behavior under pathological conditions in vitro. The specific operating conditions of each step (such as cell density, culture time, concentration of added factors, etc.) may need to be optimized according to the specific purpose of the experiment.
Table 1. Macrophage culture and induction conditions
|
Cell source |
Culture medium |
Initial Additions |
M1 polarization |
M2 polarization |
|
THP-1Cell |
RPMI 1640 |
100 ng/mL PMA |
20 ng/mL IFN-γ 100 ng/mL LPS |
20 ng/mL IL-4 20 ng/mL IL-13 |
|
Monocytes |
RPMI 1640 |
M1:50 ng/mL GM-CSF M2: 50 ng/mL M-CSF |
10 ng/mL LPS 50 ng/mL IFN-γ |
M2a: 20 ng/mL IL-4 M2b: IgG+LPS M2c: 20 ng/mL TGF-β1 or 10 ng/mL IL-10 |
|
Marrow |
IMDM |
10-50 ng/mL M-CSF |
100 ng/mL LPS (50ng/mL IFN-γ can be added) |
10 ng/mL IL-4 (10 ng/mL IL-13 can be added) |
|
RAW264.7 Cell |
DMEM |
/ |
100 ng/mL LPS |
20 ng/mL IL-4 (20 ng/mL IL-10 can be added） |
5.Assay Data
YEASEN provides a series of HiActive® high-activity cytokine products related to macrophage culture to support macrophage research.
HiActive® Highly Active Cytokines：The biological activity of each cytokine has been verified to ensure the high activity of the cytokine.
Activity verification data:
Recombinant Mouse M-CSF Protein
Figure 1. Measured in a cell proliferation assay using M‑NFS‑60 mouse myelogenous leukemia lymphoblast cells. The ED50 for this effect is typically 16.74 -25.83 ng/ml.
Recombinant Mouse GM-CSF Protein
Figure 2.The ED50 as determined by a cell proliferation assay using murine FDC-P1 cells is 2.79-12.84 pg/mL.
Recombinant Human IL-10 Protein
Figure 3. The ED50 as determined by a cell proliferation assay using murine MC/9 cells is less than 0.1 ng/mL, corresponding to a specificactivity of > 1.0×107 IU/mg.
Related Products
|
Classification |
Species |
Cat |
Specification |
|
G-CSF |
Human |
2 μg/10 μg/ 50 μg/ 100 μg |
|
|
Mouse |
2 μg/ 10 μg/50 μg/100 μg/500 μg |
||
|
M-CSF |
Human |
10 μg/100 μg/500 μg |
|
|
Mouse |
10 μg/100 μg/ 500 μg |
||
|
GM-CSF |
Human |
5 μg/50 μg/100 μg/ 500 μg |
|
|
Mouse |
10 μg/100 μg/ 500 μg |
||
|
IFN-γ |
Human |
20 μg/50 μg/ 100 μg/ 500 μg |
|
|
Mouse |
5 μg/50 μg/ 100 μg/ 500 μg |
||
|
IL-4 |
Human |
5 μg/50 μg/ 100 μg/ 500 μg |
|
|
Mouse |
5 μg/ 100 μg/ 500 μg |
||
|
IL-10 |
Human |
2 μg/ 10 μg/ 50 μg/ 100 μg/ 500 μg |
|
|
Mouse |
2 μg/ 10 μg/ 50 μg/ 100 μg/ 500 μg |
||
|
IL-13 |
Human |
2 μg/ 10 μg/ 50 μg/ 100 μg/ 500 μg |
|
|
Mouse |
2 μg/ 10 μg/ 50 μg/ 100 μg/ 500 μg |
References
[1]Atri C, Guerfali FZ, Laouini D. Role of Human Macrophage Polarization in Inflammation during Infectious Diseases. Int J Mol Sci. 2018 Jun 19;19(6):1801.