From “suspension” to “adhesion”: complete operational guidelines for THP-1 Macrophage induction

Introduction of THP-1 cells

THP-1 cells are a peripheral blood cell line derived from a patient with human acute monocytic leukemia. Since its establishment in 1980, it has become one of the most commonly used and important in vitro model cells in immunology, inflammation research, and cancer research, especially indispensable in macrophage-associated research.

1. THP-1 cells basic information

Full name: Tohoku Hospital Pediatrics-1

Source: Isolated from the peripheral blood of a 1-year-old boy with acute monocytic leukemia.

Cell type: round or oval cells growing in suspension.

Cell properties: The undifferentiated state represents a monocytic phenotype with unlimited proliferation capacity (long passages in vitro) and retains the potential for monocytic differentiation into macrophages, making it an ideal model for studying the monocyte-macrophage lineage.

2. THP-1 cell morphology

In the undifferentiated state, THP-1 cells grow in suspension, with a round or oval shape, a diameter of about 10-15μm, large and irregular nuclei, little cytoplasm, and basophilia; when differentiated into macrophages by the treatment of inducers, the cells gradually grow in adherence to the surface, and their morphology becomes pike-shaped, polygonal, or irregularly flattened, with an increase in cytoplasm and a large number of particles (e.g., lysosomes) appear, the nucleus shrinks and the chromatin is condensed, and the overall morphology is typical of mature macrophages.

3. Culture of THP-1 cells

The culture of THP-1 cells requires strict control of environmental and nutritional conditions to maintain their activity and undifferentiated state, and the core operation points are as follows:

 

Induction and differentiation of THP-1 cells into macrophages

THP-1 cells are classical models for studying macrophage function, and their undifferentiated state is monocytes grown in suspension, which can differentiate into adherent growth cells with macrophage phenotype and function after induction. In this paper, based on the commonly used PMA (phorbol ester) induction method, combined with key details and visual annotations, to provide a complete operating procedure that can be directly landed.

1. Experimental principle

THP-1 cells are normally round and grow in suspension. When stimulated with PMA, PMA activates the protein kinase C (PKC) signaling pathway, which leads to a series of cellular responses: cell cycle arrest, upregulation of cell adhesion molecule expression, changes in cell morphology (from round to irregular, pseudopodial adherent cells), and begins to express specific surface markers of macrophages (e.g., CD16, CD64, CD68). Macrophages can be further induced to differentiate into M1 and M2 macrophages in the presence of PMA.

 

Figure 1: Induction of THP-1 Macrophages

2. Experimental reagents and materials

 

3. Experimental procedures

(1) THP-1 cell pretreatment (to ensure cell state)

① Remove THP-1 cells in logarithmic growth phase from the incubator and gently blow and beat evenly (avoid damaging the cells with excessive force);

② Mix 10 μL cell suspension with 10 μL trypan blue, count the plate, and adjust the cell density to 1×10⁶ - 2×10⁶ cells/mL;

③ Inoculate the cell suspension into the culture plate/dish and adjust the density to 1 × 10⁶ cells/mL.

(2) PMA-induced treatment (key to differentiation initiation)

① Take out PMA stock solution, melt at room temperature and vortex to mix;

② Calculate the required storage volume (a final concentration of 50-100 ng/mL is usually used) according to the preset PMA working concentration, add PMA working solution to the medium inoculated with cells, and gently shake the culture plate to mix.

③ Incubate the cells in an incubator containing 5% CO₂ at 37℃ for 24 hours, and observe under a microscope whether macrophages are successfully constructed (signs of success: adherent growth, irregular morphology, enlarged cytosol, and pseudopods protruding).

④ Post-induction treatment and resting: after 24 hours of incubation, carefully suck and discard the medium containing PMA; add prewarmed PBS buffer, gently rinse the cell surface 1-2 times to completely remove residual PMA; replace with fresh complete medium free of PMA, and continue the incubation for 24 hours. This "resting" step allows cells to recover from stimulation by PMA and stabilize their M0 macrophage state, thereby responding more accurately to subsequent polarizing stimuli (e.g. LPS/IFN-γ or IL-4/IL-13).

(3) Polarization induction (further induction into M1 and M2 type macrophages)

Further induction of macrophages into M1 classically activated macrophages

 

Figure 2: Induction of M1 Macrophages

Further induction of macrophages into M2 alternatively activated macrophages

 

Figure 3: Induction of M2 Macrophages

(4) Phenotypic validation (quality control)

Successful differentiation needs to be verified by:

① Morphological differences (microscopy)

M1 macrophages: In a pro-inflammatory environment (e.g. LPS, IFN-γ stimulation), the core function of M1 is "bactericidal and pro-inflammatory" - compact cell size, dense small lysosomes maximize "degradation efficiency", short filamentous pseudopods facilitate rapid recognition of pathogens and reduce unnecessary energy expenditure on "movement/repair".

M2 macrophages: in an anti-inflammatory environment (e.g., IL-4, IL-13 stimulation), the core function of M2 is to "remove debris and promote repair" - larger cell size and well-developed plate-like pseudopods expand the "phagocytic range" (wrapping tissue debris). The abundant endoplasmic reticulum/Golgi apparatus (the reason for cytoplasmic transparency) supports the synthesis and secretion of anti-inflammatory factors (e.g., IL-10) and repair associated proteins (e.g., collagen), which contribute to the regeneration of tissue.

② Cell markers (immunohistochemical staining and flow cytometry)

Markers Secreted by M1 and M2 Macrophages

M1/M2 macrophages can be preliminarily distinguished by morphological characteristics, and markers expressed by them (such as M1's CD86, M2's CD206) can be analyzed using immunohistochemical staining or flow cytometry to more accurately determine the phenotype and functional status of macrophages.

(5) Common problems and solutions (pit avoidance guide)

Common problems	Possible causes	Solution
< 50% adherence after 24 hours of induction	PMA concentration too low; poor cell viability	Increase PMA concentration (not more than 100 ng/mL); replace the logarithmic phase cells
Cells adherence without significant change in morphology	Insufficient incubation time; PMA inactivation	Extended incubation up to 48 hours; Reformulated PMA stock
Massive cell death after differentiation	PMA concentration too high; media contamination	Decrease PMA concentration; test medium for turbidity and replace with new medium

Following this guidance, you will be able to obtain THP-1-derived macrophages stably and efficiently, providing a reliable cell model for your scientific research.

Yeasen HiActi Cytokines

Yeasen has developed a series of HiActi® cytokines specifically designed for cell culture. Strict quality control and validation of cellular functions have ensured that the products have high activity, high purity, high stability, and low endotoxin levels. After THP-1 induces differentiation into macrophages, it can further induce differentiation into M1 and M2 types of macrophages, in which the cytokines IFN-γ, IL-4, and IL-13 play an important role in this process. Yeasen Cytokines can precisely assist the differentiation of macrophages and help you to get the ideal experimental results.

Product Data

Bioactivity of human IFN-γ

Figure 4：The ED₅₀ as measured in anti-viral 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 5：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.

Bioactivity of human IL-13

Figure 6：The ED₅₀ as determined by a cell proliferation assay using human TF-1 cells is less than 1 ng/mL, corresponding to a specific activity of > 1.0 × 10⁶ IU/mg. Fully biologically active when compared to standard.

Ordering Information

Product name	Item No.	Strength
Recombinant human interferon-γ	91207ES	20μg/50μg/100μg/500μg
Recombinant human interleukin-4	90105ES	5μg/20μg/50μg/100μg/500μg
Recombinant human interleukin-13	90112ES	2μg/10μg/50μg/100μg/500μg