Overview

 

Macrophages are an essential component of the immune system, playing a crucial role in protecting the body from infections and maintaining tissue homeostasis. To study the functions and mechanisms of macrophages, scientists have developed a model that does not require the collection of tissues from patients or animals, namely the THP-1 cell line. The THP-1 cell line, originally derived from the blood of a patient with acute monocytic leukemia, is a suspension cell type with the ability to differentiate into various macrophage phenotypes, making it an ideal choice for macrophage differentiation studies. THP-1 cells can be induced to differentiate into macrophage-like cell populations through chemical treatment, cytokine stimulation, or co-culture methods.

 

Mechanism

 

Cytokine stimulation is a common method for inducing the differentiation of THP-1 cells. Cytokines such as IL-4 and IL-13 can promote the differentiation of THP-1 cells into macrophages and activate specific signaling pathways to regulate cellular functions. This method can simulate in vivo immune regulation processes and provides a convenient approach to study the functions of macrophages in inflammation, immune regulation, and anti-tumor responses.

 

Macrophages are classified into two main types: M1 macrophages and M2 macrophages. THP-1 cells are typically induced to differentiate into macrophages using Phorbol 12-myristate 13-acetate (PMA) , and in the presence of PMA:

Lipopolysaccharide (LPS) (induction concentration: 100ng/mL) , IFN-γ (induction concentration: 20ng/mL) (abs04123) are added for 48h to induce M1 polarization;

IL-4 (induction concentration: 20ng/mL) (abs04698), IL-13 (induction concentration: 20ng/mL) (abs04079) are added for 48h to induce M2 polarization;

Finally, in the absence of stimuli and PMA, cells are resuspended in serum-free RPMI-1640 basal medium to maintain viability for 72h.

 

Induction Protocol

 

Protocol for inducing THP-1 cells to differentiate into macrophages:

(1) Culture of THP-1 cells: For more culture tips, visit THP-1 Cell Culture Guide

 

Basic Information of THP-1 (Human Monocytic Leukemia Cells)

Morphology	Lymphocyte-like
Growth Characteristics	Suspension
Nutrient System	89% RPMI1640 basal medium (abs9484) + 10% Fetal Bovine Serum (Premium) (abs972) + 1% Penicillin-Streptomycin Solution (abs9244) + 0.1% β-Mercaptoethanol Solution (abs9592)

 

THP-1 (Human Monocytic Leukemia Cells)

 

(2) Preparation of Induction Medium: Add PMA (induction concentration: 100ng/mL) to complete RPMI-1640 medium (containing fetal bovine serum) to prepare the induction medium;

(3) Cell Seeding: Collect THP-1 cells, count them, and resuspend the cell pellet in induction medium to adjust the cell density to 5*10^5/mL. Seed the cells into a 6-well plate, adding 2mL of cell suspension per well;

(4) Induction Culture: After 48h, observe the successful construction of macrophages under a microscope;

(5) Signs of Successful Induction: After PMA induction, THP-1 cells change from suspension growth to adherent growth, from round to irregular shapes, with increased cell volume, loose cytoplasm, enlarged nuclei, and visible organelles, with small protrusions visible around the cell membrane.

 

Phase-contrast micrograph of PMA-induced THP-1 cells

 

THP-1 cells (40X) before (A) and after (B) 48h of PMA treatment show morphological changes from round to elongated and flattened shapes.

 

Protocol for further inducing macrophages into M1 phenotype:

(1) Preparation of Induction Medium: Add PMA (induction concentration: 100ng/mL), Lipopolysaccharide (LPS) (induction concentration: 100ng/mL), and IFN-γ (induction concentration: 20ng/mL) (abs04123) to complete RPMI-1640 medium (containing fetal bovine serum) to prepare the induction medium;

(2) Induction Culture: Remove the supernatant of pre-induced macrophages and add the induction medium (2mL per well in a 6-well plate) for 48h to induce M1 polarization. After 48h, observe the successful construction of M1 cells under a microscope;

(3) Finally, in the absence of stimuli and PMA, resuspend cells in serum-free RPMI-1640 basal medium to maintain viability for 72h.

 

Protocol for further inducing macrophages into M2 phenotype:

(1) Preparation of Induction Medium: Add PMA (induction concentration: 100ng/mL), IL-4 (induction concentration: 20ng/mL) (abs04698), and IL-13 (induction concentration: 20ng/mL) (abs04079) to complete RPMI-1640 medium (containing fetal bovine serum) to prepare the induction medium;

(2) Induction Culture: Remove the supernatant of pre-induced macrophages and add the induction medium (2mL per well in a 6-well plate) for 48h to induce M2 polarization. After 48h, observe the successful construction of M2 cells under a microscope;

(3) Finally, in the absence of stimuli and PMA, resuspend cells in serum-free RPMI-1640 basal medium to maintain viability for 72h.

 

Illustration of THP-1 polarization into M1 and M2 macrophages

 

M1 macrophages exhibit diverse morphologies with numerous pseudopodia and clear branching, while M2 macrophages have uniform elongated shapes with less branching.

 

Identification of M1 and M2 Macrophages

 

The above morphological characteristics are preliminary identification methods. More accurate identification can be achieved through flow cytometry, which typically involves macrophage-specific markers. Common M1 macrophage markers include CD68, CD80, CD86, CD32, etc.; common M2 macrophage markers include CD206, CD204, CD163, etc.

 

Catalog Number	Product Name	Specification
abs04123	Recombinant Human IFN-γ Protein	50ug/100ug/500ug/1mg
abs04698	Recombinant Human IL-4 Protein	10ug/100ug/500ug
abs04079	Recombinant Human IL-13 Protein(C-6His)	10ug/50ug/500ug/1mg

 

Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us

Absin Bioscience Inc. Email: worldwide@absin.cn

Follow us on Facebook: Absin Bio