I. Macrophages

1. Origins of Macrophages

1) In Vivo Natural Sources

• The primary source is hematopoietic stem cells in the bone marrow, which differentiate into monocytes, migrate to tissues, and mature into macrophages.
• Some embryonic-derived macrophages (e.g., microglia) settle in tissues during the embryonic period and self-renew through local proliferation.

2) In Vitro Experimental Sources

• Bone marrow-derived: Hematopoietic stem cells/monocytes are isolated from bone marrow and induced to differentiate in vitro into bone marrow-derived macrophages (BMDMs), serving as a classic model for macrophage research.
• Monocytes from PBMC: Monocytes isolated from PBMC can differentiate into macrophages in vitro under M-CSF/GM-CSF induction, representing a commonly used laboratory method for acquisition.

2. Functional Phenotype Classification of Macrophages

1) M1 Macrophages (Pro-Inflammatory): Activated by IFN-γ, LPS, etc., primarily secrete pro-inflammatory factors like TNF-α and IL-6. Responsible for phagocytic clearance of pathogens, participating in immune defense and inflammation initiation.

2) M2 Macrophages (Anti-Inflammatory/Repair Type): Induced by IL-4, IL-13, IL-10, etc., they are subdivided into M2a, M2b, M2c subtypes. Their core function is to suppress excessive inflammation, promote tissue repair and vascular regeneration, and maintain tissue homeostasis.

Figure 1. Classification of Macrophages

3. Functions of Macrophages

Macrophages are multifunctional, versatile immune cells whose roles span daily homeostasis to acute infection response, from promoting repair to regulating immune balance—encompassing all facets of life processes. Their proper functioning is vital for health, while dysfunction is closely linked to infections, autoimmune diseases, cancer, metabolic disorders, and other conditions.

Core Functions	Specific Roles
Immune Defense	Phagocytose and kill pathogens, initiate adaptive immunity, recruit immune cells
Tissue Homeostasis	Clear apoptotic cells, promote tissue repair, regulate metabolism
Immune Regulation	Initiating inflammatory responses, terminating inflammation, immunosuppression
Specialized Functions	Bone resorption (osteoclasts), neural pruning (microglia), blood filtration (Kupffer cells)

II. Bone marrow-derived macrophages

BMDMs refer to macrophages differentiated from hematopoietic progenitor cells extracted from mouse (most commonly) or other animal bone marrow. This differentiation occurs in vitro over approximately 7 days under the induction of specific cytokines, primarily macrophage colony-stimulating factor (M-CSF).

The selection of bone marrow-derived macrophages is driven by the following advantages:

Research Requirements	Advantages of BMDMs
High Cell Yield	Large quantities of highly proliferative cells can be obtained from a small number of mice
Ensuring Experimental Purity	Homogeneous cell populations with purity exceeding 95%
Achieves reproducibility	Controllable culture conditions minimize batch-to-batch variability
Facilitates mechanism exploration	Facilitates genetic manipulation and precise spatiotemporal stimulation
Simulation of disease states	Can be induced into various disease-associated phenotypes such as M1, M2, and TAMs

III. Culture of Bone marrow-derived macrophages

Figure 2. Scheme for the isolation, formation, and stimulation of mouse BMDMs[1]

1. Isolation of mouse bone marrow cells

1) Isolation of Femur and Tibia: Take 6-8 weeks-old mice, isolate the femur and tibia, rinse off hair, then trim both ends of the bones.

2) Wash Bone Marrow: Using a needle and syringe, wash the bone marrow into pre-chilled PBS + 2% heat-inactivated fetal bovine serum (FBS).

3) Disperse Cells by Pipetting: Pipette the bone marrow 4–6 times through the needle to disperse the cells.

4) Filter Cell Suspension: Pass the cell suspension through a 70 μm cell strainer to remove cell clumps, bone debris, hair, and other cellular/tissue fragments.

5) Red Blood Cell Lysis: Add 3× volume of NH₄Cl solution (0.8% NH₄Cl solution) and incubate on ice for 10 minutes to remove red blood cells.

6) Cell Centrifugation: Centrifuge at 4°C, 500 × g for 5 minutes.

7) Resuspend Cells: Resuspend the cell pellet in pre-chilled PBS + 2% FBS (20–50 mL, depending on cell number).

2. Induction of bone marrow-derived macrophages

1) Resuspend Cells: Resuspend isolated bone marrow cells in BMDM growth medium (IMDM medium + 10% FBS + 10 ng/mL recombinant M-CSF) at a concentration of 2 × 10⁶ cells/mL.

2) Seed Cells: Seed cells into 6-well or 12-well tissue culture plates.

3) Media Change: Replace with fresh BMDM growth medium on day 3.

4) Maturity Assessment: On day 7, assess mature BMDM formation by flow cytometric analysis using fluorescently labeled antibodies to detect cells expressing CD11b, and F4/80.

3. Polarization Induction of Bone Marrow-Derived Macrophages

On day 7 of culture, replace with fresh stimulatory medium.

• For M1 Activation: Use IMDM medium containing 10% FBS and 100 ng/mL LPS, or IMDM medium containing 100 ng/mL LPS and 50 ng/mL IFN-γ.
• For M2 Activation: Use IMDM medium containing 10% FBS and either 10 ng/mL IL-4 or 10 ng/mL IL-13.

4. Macrophage Identification

1) Cell Surface Antigen Expression Detection

Detect cell surface antigen expression at different time points using antibodies following standard flow cytometry staining protocols.

• Characteristic markers for M1 macrophages: CD11c, CD80, CD86.
• Characteristic marker for M2 macrophages: CD206.

2) Detection of Activation-Related Genes and Signaling Pathways

• qRT-PCR was used to detect the expression of activation-related genes in M1 and M2 macrophages.
  • M1 activation-specific genes: IL-1β, TNF-α, IL-6.
  • M2 activation-specific genes: IL-10, IL-13, Arginase 1, PPARγ.
• Western Blot analysis was used to assess the activation status of cellular signaling pathways involved in M1 or M2 macrophage activation.

IV. Analysis of Core Cytokine Functions

The core role of cytokines in bone marrow-derived macrophage (BMDM) culture is to drive differentiation and regulate phenotype. M-CSF/GM-CSF primarily governs maturation, while IFN-γ, IL-4, and others regulate functional polarization, collectively determining BMDM functional characteristics.

1. Core Cytokines Driving Differentiation (Essential for In Vitro Culture)

1) Macrophage Colony-Stimulating Factor (M-CSF)

• Core Function: The "gold standard" factor for BMDM differentiation, specifically driving the directed maturation of hematopoietic stem/progenitor cells and monocytes into mature macrophages.
• Key Effects: Promotes cell proliferation, inhibits apoptosis, and induces maturation of core macrophage functions (phagocytosis, cytokine secretion). Differentiated BMDMs exhibit uniform phenotypes and stable functions, making it the preferred induction factor for basic research.

2) Granulocyte-macrophage colony-stimulating factor (GM-CSF)

• Core Function: A differentiation inducer that replaces M-CSF, possessing the potential to drive both macrophage and granulocyte differentiation.
• Key Effects: Differentiated BMDMs exhibit a more pronounced "inflammatory macrophage" phenotype with enhanced antigen-presenting capacity. Frequently used in studies requiring amplified immune responses (e.g., anti-infection, tumor immunity).

2. Key cytokines regulating phenotypic polarization (post-differentiation activation)

1) M1-Polarization-Related Factors (Pro-inflammatory Phenotype)

• Core Combination: Lipopolysaccharide (LPS) + Interferon-γ (IFN-γ)
• Mechanism of Action: LPS activates the TLR4 signaling pathway to initiate the inflammatory response, while IFN-γ amplifies pro-inflammatory effects, jointly inducing M1 polarization of BMDMs.
• Key Effects: High expression of markers like CD80, CD86, and CD11c; massive secretion of proinflammatory cytokines including IL-1β, TNF-α, and IL-6; enhanced pathogen clearance and immune defense functions.

2) M2-Polarization-Related Factors (Anti-inflammatory/Repair Phenotype)

• Core Factors: Interleukin-4 (IL-4), Interleukin-13 (IL-13)
• Mechanism of Action: Induces M2 polarization of BMDMs by activating the JAK-STAT6 signaling pathway; can be used alone or in combination.
• Key Effects: Highly express markers like CD206, secrete anti-inflammatory factors such as IL-10 and IL-13, activate Arginase 1, suppress excessive inflammation, promote tissue repair, and maintain metabolic homeostasis.

3. Auxiliary Regulatory Factors (Optimizing Phenotype or Enhancing Effects)

1) Lipopolysaccharide (LPS)

• Core Function: "Initiator" for M1 polarization, often combined with IFN-γ to enhance pro-inflammatory effects.
• Key Effects: Directly activates innate immune signaling in macrophages, rapidly induces pro-inflammatory factor secretion, and mimics the in vivo infection or inflammatory microenvironment.

2) Interleukin-10 (IL-10)

• Core Function: "Reinforcing factor" for M2 polarization, suppressing M1 phenotype and promoting M2 stability.
• Key Effects: Downregulates pro-inflammatory factor expression while upregulating anti-inflammatory genes (e.g., IL-10 itself, Arginase 1), maintaining the anti-inflammatory state of macrophages to prevent tissue damage.

V. Yeasen HiActi™ Cytokines

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Product Data

Bioactivity of Mouse M-CSF

Figure 3: Measured in a cell proliferation assay using MNFS60 mouse myelogenous leukemia lymphoblast cells. The EC50 for this effect is 0.21.5 ng/mL.

Bioactivity of Mouse IFN-γ

Figure 4: Measured in an anti-viral assay using L-929 mouse fibroblast cells infected with encephalomyocarditis (EMC) virus. The EC₅₀ for this effect is 0.03-0.1 ng/mL.

Bioactivity of Mouse IL-4

Figure 5: Measured in a cell proliferation assay using HT-2 mouse T cells. The EC₅₀ for this effect is 0.2–1.0 ng/mL.

Ordering Information

Product Name	Cat. No.
Recombinant Mouse M-CSF/CSF1 Protein (HEK293)	92116ES
Recombinant Mouse GM-CSF Protein	91108ES
Recombinant Mouse IFN-gamma Protein	91212ES
Recombinant Mouse IL-4 Protein	90144ES
Recombinant Mouse IL-10 Protein	90149ES
Recombinant Mouse IL-13 Protein	90151ES

References

1. Zhou B, Safe SH, Bazer FW, Cheruku PS, Ying W. Investigation of Macrophage Polarization Using Bone Marrow-Derived Macrophages. 2013; (76).