Macrophage Differentiation and Cytokine Profiling

Human leukemia monocytic cells of the THP-1 cell line (TIB-202 ^TM; ATCC, Manassas, VA, USA) were cultured in RPMI 1640 (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 100 U/mL penicillin, 100 µg/mL streptomycin (Invitrogen), and 50 µM 2-mercaptoethanol. After that, THP-1 cells were incubated with 100 ng/mL of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for macrophage differentiation [30 (link)]. Then, the differentiated THP-1 cells were stimulated with RPMI (untreated), LPS from E. coli 026:B6 (100 ng/mL) (Sigma-Aldrich), interleukin 4 (IL-4) (20 ng/mL), and 20% tumor-conditioned media prepared by centrifuging 5 × 10⁶ CaCO2 and HepG2 cells (cancer cell lines) incubated in the presence or absence of BAM15 (50 ng/mL) [31 (link)]. At 24 h after stimulation, supernatants were collected for cytokine quantification using ELISA (Invitrogen), and the cells were harvested for quantitative real-time polymerase chain reaction (PCR).