Preparation before polarization:

The passage number of cells used for polarization induction should not be too high. Before inducing polarization, use flow cytometry to detectTHP-1cellCD14andCD11bexpression. OnlyTHP-1cells that do not express or have low expression of both markers can be used for subsequent experiments.

M1/M2Induction

1.M1Induction:

UseM0induction medium (1640complete medium containing100 ng/mL PMA), seedTHP-1>95%,<20passages) at a density of 5×10⁵ cells/mL into10 cm culture dishes (10 mL), culture for24h to generate macrophagesM0 (cells adhere, pseudopodia extend). Gently wash with pre-warmedPBSfor1-2times to removePMA. Replace withM1induction medium (1640complete medium containing100 ng/mL LPS,20 ng/mL IFN-γ), culture for48h.

Negative control: UseM0induction medium (1640complete medium containing100 ng/mL PMA), seedTHP-1>95%,<20passages) at a density of 5×10⁵ cells/mL into10 cm culture dishes (10 mL), culture for24h to generate macrophagesM0 (cells adhere, pseudopodia extend). Gently wash with pre-warmedPBSfor1-2times to removePMA. Replace with complete medium and culture for48h. It is recommended to useAccutasefor sample collection and testing.

2.M2Induction:

UseM0induction medium (1640complete medium containing100 ng/mL PMA), seedTHP-1>95%,<20passages) at a density of 5×10⁵ cells/mL into10 cm culture dishes (10 mL), culture for24h to generate macrophagesM0 (cells adhere, pseudopodia extend). Gently wash with pre-warmedPBSfor1-2times to removePMA, replace with complete medium and rest for24h. Then replace withM2induction medium (1640complete medium containing20 ng/mL IL-4,20 ng/mL IL-13), culture for24h.

Negative control: UseM0induction medium (1640complete medium containing100 ng/mL PMA), seedTHP-1>95%,<20passages) at a density of 5×10⁵ cells/mL into10 cm culture dishes (10 mL), culture for24h to generate macrophagesM0 (cells adhere, pseudopodia extend). Gently wash with pre-warmedPBSfor1-2times to removePMA, replace with complete medium and rest for48h. It is recommended to useAccutasefor sample collection and testing.

Cat No.	Product name	Reactivity	ExpSys	Quantity
UA040053	IFN-γ Protein, Human	Human	E.coli	1
UA040467	IL-13 Protein, Human	Human	E.coli	1
UA040026	IL-4 Protein, Human	Human	E.coli	1
UA080443	Lipopolysaccharide (LPS) solution (500X)			1

Human THP-1 Cell Polarization Induction Kit