Macrophage Polarization and Co-culture

THP-1 (Human acute monocytic leukemia cell line) cells and L02 cells were purchased from ATCC. The cells were all cultured in RPMI 1640 containing 10% heat-inactivated FBS and 1% penicillin–streptomycin in a 5% CO₂ atmosphere at 37 °C. THP-1 monocytes were differentiated into M0-like macrophages by incubation with 160 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma, P8139) for 48 h in RPMI medium. PMA-induced macrophages were polarized into M1 macrophages by incubation with 20 ng/ml of IFN-γ (R&D system, #285-IF) and 10 pg/ml of LPS (Sigma, #8630). Macrophage M2 polarization was obtained by incubation with 20 ng/ml of interleukin 4 (R&D system, #204-IL) and interleukin 13 (R&D system, #213-ILB). In the co-culture experiments, 0.5 × 10⁶ THP-1 monocytes were cultivated and differentiated for 48 h before being incubated respectively with 0.5 × 10⁶ L02-SCR or L02-Sh cells in 6-well Transwell plates for 48 h^{37 (link)} .