Phorbol myristate acetate (pma)

The human monocytic cell line THP-1 was purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 medium with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37 °C in an incubator with a humidified atmosphere containing 5% CO₂.
1 × 10⁶ THP-1 cells were seeded into 6-well plate and differentiated with PMA (Sigma-Aldrich, St Louis, MO, USA) for 48 h in a concentration of 50ng/ml. Cell adhesion, spreading and decreased nucleocytoplasmic ratio, as the main features for macrophage differentiation, were visualized by phase-contrast microscopy. Surface markers of CD14 (monocyte marker) and CD68 (macrophage marker) during differentiation were detected by flow cytometry. To measure the response of PMA-differentiated macrophages to LPS (Escherichia coli 055:B5; Sigma-Aldrich, St Louis, MO, USA), cells were exposed to LPS (1 µg /ml) for 12 h after a recovery period (an incubation of 12 h in fresh RPM-1640 removing PMA and 10% FBS) and levels of TNFα in cell supernatants were quantified using Human TNFα ELISA Kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. After the acquisition of a macrophage-like phenotype, cells were treated with LPS (1 µg /ml) for 0, 1, 3, 6, 12 and 18 h, respectively, and macrophage model of LPS-induced ARDS was successfully established.

We treated THP-1 monocytes with 100 ng/ml phorbol 12-myristate 13-acetate (PMA, Sangon Biotech, China) for 48 h followed by 20 ng/ml IL-4 (Peprotech, USA) for 24 h to obtain M2-polarized TAMs. Then, the THP-1 cells were further cultured in serum-free RPMI-1640 medium for 24 h. The culture supernatants were collected, centrifuged at 300 xg for 5 minutes, filtered through a 0.22 μm filter mesh, and stored at -80° C as TAM conditioned medium (TAM-CM).