I. Peripheral Blood Mononuclear Cells (PBMCs)

1. Peripheral Blood

Core Definition

Peripheral Blood refers to blood circulating throughout the body's circulatory system, excluding bone marrow. It contrasts with "bone marrow blood." Routine blood tests (e.g., fingerstick blood, venous blood draws) obtain peripheral blood.

Primary Components

Peripheral blood consists of plasma and blood cells. Plasma, the liquid component of blood, accounts for approximately 55% of peripheral blood volume. Its primary component is water, supplemented by electrolytes, hormones, albumin, coagulation proteins, and other substances. Blood cells include red blood cells, white blood cells, and platelets. Red blood cells transport oxygen and carbon dioxide, white blood cells participate in immune defense against pathogens, and platelets play a crucial role in hemostasis and blood clotting. Additionally, peripheral blood contains a very small amount of hematopoietic stem cells, approximately 0.01%.

2. Peripheral Blood Mononuclear Cells (PBMC)

Core Definition and Essence

1) Source: Peripheral blood (arterial, venous, capillary circulating blood), isolated from whole blood via density gradient centrifugation (Ficoll-Paque method).

2) Core Characteristics: Contains only cells with a single nucleus, excluding multinucleated cells (granulocytes) and anucleated cells (red blood cells, platelets).

3) Essence: The "core cellular reservoir" of the body's immune system, performing critical functions such as immune defense and immune regulation.

Primary Cell Composition

Cell Types	Percentage	Core Subpopulations	Key Functions
Lymphocytes	70%–90%	T cells B cells Natural Killer (NK) Cells	T cells: Cellular immunity, immune regulation B cells: Humoral immunity NK cells: Innate immunity
Monocytes	10%–30%	Classic type (CD14⁺CD16⁻) Intermediate type (CD14⁺CD16⁺) Non-classical (CD14⁻CD16⁺)	Migrate to tissues, differentiate into macrophages/dendritic cells (DCs), participate in phagocytosis, antigen presentation, and inflammatory regulation
Dendritic cells (DC)	1%–5%	Myeloid DC (mDC) Plasmacytoid DC (pDC)	Antigen-presenting cells bridging innate and adaptive immunity (activating T cells)

II. PBMC-derived macrophages

From monocytes to macrophages, this process occurs naturally within the human body:

1. monocytes are produced in the bone marrow and then released into the peripheral blood (thus, monocytes are one of the components of PBMCs).

2. Circulating through the bloodstream, monocytes traverse the vascular wall into tissues upon detecting signals of inflammation or tissue injury.

3. Within specific tissue environments, monocytes differentiate and mature into macrophages. Macrophages are the "scavengers" and immune regulators that reside within tissues, possessing more potent functions.

In the laboratory, we simulate this process:

PBMCs (isolated from peripheral blood) → Monocyte extraction → In vitro culture + Cytokine stimulation → Differentiation into macrophages

III. PBMC-derived macrophage culture

Phase 1: Isolation of PBMCs from peripheral blood

Material Preparation: Peripheral blood from healthy volunteers (collected using heparin sodium or EDTA anticoagulant tubes); lymphocyte separation media such as Ficoll-Paque PLUS; D-PBS (Dullery phosphate-buffered saline) without calcium and magnesium ions; RPMI-1640 or DMEM complete medium (basal medium + 10% fetal bovine serum + 1% penicillin/streptomycin).

Procedure:

1. Dilute blood with an equal volume of D-PBS.

2. Carefully add the diluted blood onto the surface of the pre-layered Ficoll solution, maintaining a clear interface.

3. Centrifugation: Centrifuge at 400 g for 30-35 minutes at room temperature.

4. After centrifugation, the liquid separates into multiple layers. Carefully aspirate the central white cloudy layer (i.e., the PBMC layer) with a pipette and transfer it to a new centrifuge tube.

5. Wash cells 2-3 times with PBS (centrifuge at 250-300 g for 10 minutes) to remove residual platelets and Ficoll.

6. Resuspend cells in an appropriate volume of complete medium and perform cell counting.

Stage 2: Monocyte Enrichment

After obtaining PBMCs, monocytes must be enriched from them. The adhesion method is the most commonly used and economical approach.

Principle: Monocytes exhibit strong adhesion capacity, whereas lymphocytes have poor adhesion. Short-term culture enables separation of monocytes from lymphocytes.

Procedure:

1. Add the resuspended PBMC suspension to cell culture flasks or culture plates.

2. Incubate at 37°C with 5% CO₂ for 1-2 hours to allow monocytes to fully adhere.

3. After incubation, gently swirl the culture vessel and aspirate the supernatant. The supernatant primarily contains non-adherent lymphocytes (T cells, B cells, NK cells, etc.).

4. Add pre-warmed D-PBS to the culture vessel, gently swirl, and aspirate to wash away unattached or weakly adherent cells. Repeat this step 2-3 times.

5. After washing, the cells remaining at the bottom of the flask/plate are highly purified monocytes.

Stage 3: Induction of Macrophage Differentiation

This is the most critical step, converting monocytes into mature macrophages.

Induction Medium:

• Base Medium: RPMI-1640 or DMEM complete medium.
• Key Additive: Macrophage Colony-Stimulating Factor (M-CSF): This is the core factor for inducing differentiation.
• M-CSF working concentration: Typically 20-50 ng/mL.

Procedure:

1. Add complete medium containing M-CSF to culture dishes containing adherent monocytes.

2. Incubate at 37°C in a 5% CO₂ incubator.

3. Differentiation: Typically requires 5 to 7 days.

4. Morphological Changes: Observed under an inverted microscope, cells gradually enlarge and elongate over the first few days, transforming from round to irregular shapes and extending pseudopodia, ultimately forming the characteristic macrophage morphology (pavement-like).

5. Medium change: Typically perform a 50% medium change on day 3 or 4 of culture, replenishing with fresh M-CSF-containing medium to provide nutrients and growth factors.

Key Considerations

1. Aseptic Operation: The entire process must be performed in a laminar flow hood to strictly prevent microbial contamination.

2. Cytokine Selection:

• M-CSF: Induces macrophages with a more stable, anti-inflammatory, and reparative function.
• GM-CSF: If the research objective is to induce macrophages associated with inflammation or antitumor activity, GM-CSF can be used (typically at a concentration of 20-50 ng/mL). GM-CSF induces cells more similar to dendritic cells or inflammatory macrophages.

3. FBS Bath: Fetal bovine serum (FBS) batches significantly impact cell growth. It is recommended to pre-screen and stockpile high-quality serum from the same batch.

4. Cell Characterization: After differentiation, assess purity and phenotype by flow cytometry for macrophage-specific markers (e.g., high CD14 expression, CD68, CD11b, CD163).

5. Polarization Induction: After obtaining unpolarized macrophages, stimulate them with different cytokines for 24-48 hours based on experimental requirements to induce polarization toward specific phenotypes:

• M1 type (pro-inflammatory): IFN-γ (20 ng/mL) + LPS (100 ng/mL).
• M2 type (anti-inflammatory): IL-4 (20 ng/mL) + IL-13 (20 ng/mL).

IV. Analysis of Core Cytokine Actions

In the culture of PBMC-derived macrophages, core cytokines are primarily divided into two categories: one promotes the differentiation of monocytes into mature macrophages (M0 type), while the other further polarizes M0 type macrophages into functionally distinct M1 or M2 subtypes. Different cytokines exert unique effects by regulating cellular phenotypes and signaling pathways.

Differentiation of Monocytes into Mature Macrophages

Macrophage colony-stimulating factor (M-CSF), also known as CSF-1, is a key factor in the differentiation of monocytes into mature M0-type macrophages. It regulates monocyte survival, proliferation, and differentiation, inducing gradual transformation into enlarged, adherent mature macrophages while enhancing phagocytic capacity—such as uptake of platelets and dextran. Additionally, this factor promotes the high expression of phenotypic molecules such as CD14 and CD163 in macrophages, laying the foundation for subsequent polarization toward the M2 type.

Granulocyte-macrophage colony-stimulating factor (GM-CSF): It induces the differentiation of monocytes in peripheral blood mononuclear cells (PBMCs) into macrophages and exhibits pro-inflammatory tendencies. Unlike M-CSF, it induces macrophages with low expression of M2-associated markers like CD14 and CD163, while promoting high expression of antigen-presenting molecules such as HLA-DR. These macrophages subsequently polarize more readily toward the M1 phenotype. During differentiation, GM-CSF enhances macrophage antigen-presenting capacity, preparing the body to initiate an inflammatory response.

Interleukin-34 (IL-34): Serves as an alternative factor to M-CSF, inducing monocyte differentiation into macrophages with effects similar to M-CSF. Macrophages induced by IL-34 more closely resemble the true tumor-associated macrophage phenotype in vivo, granting it unique advantages in simulated tumor-associated macrophage culture and drug screening experiments.

Macrophage Polarization

Core Factors for M1 Polarization: These factors primarily induce pro-inflammatory characteristics in macrophages, enhancing their anti-infective and anti-tumor capabilities. Among them, interferon-γ (IFN-γ) serves as a core initiator, activating inflammatory signaling pathways within macrophages. This significantly boosts their ability to kill pathogens and tumor cells while upregulating the expression of pro-inflammatory molecules such as CD86 and MHC-II. Lipopolysaccharide (LPS, though not a cytokine, often synergizes with cytokines) used in combination with IFN-γ produces a synergistic effect, further amplifying the pro-inflammatory response of macrophages. This includes the massive secretion of inflammatory cytokines such as TNF-α and IL-1, substantially enhancing the immune activation function of macrophages.

Core M2-Polarizing Factors: These factors redirect macrophages toward anti-inflammatory and tissue-repair functions while reducing inflammatory factor release. Interleukin-4 (IL-4) and Interleukin-13 (IL-13) form a classic combination. Their synergistic action induces high expression of M2-marker molecules like CD206 and CD163 in macrophages while suppressing pro-inflammatory factor secretion, thereby promoting macrophage participation in post-injury tissue repair processes such as extracellular matrix synthesis. Interleukin-10 (IL-10) primarily enhances the anti-inflammatory effects of macrophages. It further suppresses M1 macrophage-associated inflammatory responses, maintains the immune tolerance state of macrophages, and plays a crucial role in alleviating inflammatory damage and maintaining internal environmental stability.

V. Yeasen HiActi™ Cytokines

Recombinant proteins have been widely applied in core fields such as stem cell and organoid culture, recombinant protein therapeutics, CAR-T cell therapy, and antibody drugs. With the rapid growth of the biopharmaceutical industry, the recombinant protein market is expanding rapidly, and demand for high-end raw materials is increasing year by year. To precisely meet the continuously upgrading application needs in both research and industrial settings, addressing key pain points such as low protein activity and insufficient batch-to-batch stability, Yeasen Biotech leverages its years of R&D and production experience and technological accumulation at and to build an innovative recombinant protein expression and purification platform, focusing on providing high-activity recombinant protein products. Leveraging its proprietary expression and purification platform, Yeasen Biotech has developed HiActi™ cytokines including M-CSF, GM-CSF, and IFN-γ. These products undergo rigorous quality control and cellular function validation to ensure high activity, purity, stability, and low endotoxin levels, helping you achieve optimal experimental results.

Product Data

Bioactivity of Human M-CSF 

Figure 1. Measured in a cell proliferation assay using M-NFS-60 mouse myelogenous leukemia lymphoblast cells. The EC₅₀ for this effect is 0.2–1.5 ng/mL.

Bioactivity of Human GM-CSF

Figure 2. The ED₅₀ as determined by a cell proliferation assay using human TF-1 cells is less than 0.1 ng/mL, corresponding to a specific activity of > 1.0 × 107 IU/mg.

Bioactivity of Human IFN-γ

Figure 3. The ED₅₀ as measured in antiviral assays using human HeLa cells infected with encephalomyocarditis (EMC) virus is 0.15–0.80 ng/mL. Fully biologically active when compared to standard.

Bioactivity of human IL-4

Figure 4. The ED50 as determined by a cell proliferation assay using human TF-1 cells is less than 0.2 ng/mL, corresponding to a specific activity of > 5.0 ×10⁶ IU/mg. Fully biologically active when compared to standard.

Ordering Information

Product Name	Cat. No.
Recombinant Human IFN-γ Protein	91207ES
Recombinant Human IL-4 Protein	90105ES
Recombinant Human IL-13 Protein	90112ES
Recombinant Human M-CSF Protein	91103ES
Recombinant Human GM-CSF Protein	91102ES
Recombinant Human IL-10 Protein	90109ES
Recombinant Human IL-34 Protein	90206ES