*3.6. Macrophage Polarization*

THP1 cells were maintained in RPMI 1640 (Gibco, Grand Island, NY, USA) with a supplement of 10% FBS (Gibco) and 100 U/mL penicillin/streptomycin (Gibco). To induce monocytic differentiation, THP1 cells were plated at a density of 4 <sup>×</sup> <sup>10</sup><sup>5</sup> /well on 6-well culture plate and pre-treated with PMA (Sigma) for 48 h. After PMA treatment, cells were stabilized for another 24 h in the maintenance

media. Macrophages were then polarized in M1 macrophages by incubation with 20 ng/mL of IFN-γ (Peprotech) and 10 μg/mL of LPS (Sigma) for 5 days. PMA-pretreated cells were cultured within the maintenance media for 5 days without any lineage-specific stimulants for the spontaneous polarization towards M2 macrophages. To assess the Ech A impact on macrophage polarization, Ech A or vehicle was also treated during the culture period. Five days later, the culture supernatant was collected and secreted cytokine levels were estimated using human TNF-α and the IL-10 Duoset ELISA kit (R&D system, Abingdon, UK).
