Cell culture and THP-1 differentiation

Human monocytic THP-1 cells (TIB-202) was obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in RPMI 1640 medium (HyClone™, Utah, USA) containing 10% (v/v) fetal bovine serum (Gibco, South America), 10 mM 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid (HEPES; HyClone™), 2 mM L-glutamine (Gibco), 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco) and 0.05 mM 2-mercaptoethanol (Bio-Rad) at 37˚C in a humidified atmosphere of 5% CO₂. THP-1 cells (1×10⁵ cells/well in 96-well tissue culture plates (Nunc™, Roskilde, Denmark) or 2×10⁶ cells/well in 6-well plates (Nunc™)) were differentiated into macrophages by incubating with 50 ng/mL PMA for 24 h at 37 ^OC in 5% CO₂. After washing to remove non-adherent cells, the adherent macrophages were incubated in antibiotic- and FBS-free RPMI for 1 h at 37 ^OC in 5% CO₂ and used in subsequent experiments.