Differentiation of THP1 Cells into TAM Model

The in vitro TAM model was generated as follows. Briefly, THP1 cells (5 × 10⁵ cells/mL) were differentiated using 200 nM phorbol 12-myristate 13-acetate (PMA) (32 (link)) (Solarbio, Cat. no.: p6741) for different time-periods (0, 24, 48, 72, and 96 h); we observed the changes in cellular morphology under an inverted phase-contrast microscope. Differentiation of PMA-treated cells was enhanced after the initial 3-d impetus by removing the PMA-containing medium and then incubating the cells in fresh RPMI 1640 with 20 ng/mL recombinant human IL-4 (PeproTech, Cat. no.: 200-04] and 20 ng/mL recombinant human IL-13 (PeproTech, Cat. no.: 200-13) for different time-periods (0, 24, 48, and 72 h) The complete medium was then replaced and cultured for another 2 days, and the supernatant was collected for subsequent experiments.