Macrophage polarization in co-culture

Human monocytic THP-1 cells were maintained in culture in Roswell Park Memorial Institute medium (RPMI 1640, Invitrogen) culture medium containing 10 % of heat inactivated fetal bovine serum (Invitrogen) and supplemented with 10 mM Hepes (Gibco, #15630-056), 1 mM pyruvate (Gibco, #11360-039), 2.5 g/l D-glucose (Merck) and 50 pM ß-mercaptoethanol (Gibco; 31350–010). THP-1 monocytes are differentiated into macrophages by 24 h incubation with 150 nM phorbol 12-myristate 13-acetate (PMA, Sigma, P8139) followed by 24 h incubation in RPMI medium. Macrophages were polarized in 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 Systems, #204-IL) and 20 ng/ml of interleukin 13 (R&D Systems, #213-ILB). HepG2 and A549 cells were respectively cultivated in Dulbecco’s modified Eagle's minimal essential medium (DMEM medium 1 g glucose/l) (Gibco) and Minimum Essential Medium Eagle medium (MEM) (Gibco), both containing 10 % fetal bovine serum. In the co-culture experiments, THP-1 monocytes were differentiated in 6 Transwell inserts (membrane pore size of 0.4 μm, Corning, #3450). Macrophages and HepG2 cells were co-cultured in CO₂ independent medium supplemented with 0.5 mM L-glutamine (Sigma, # G3126) and 3.75 g/l of D-glucose (Sigma, #50-99-7) for 16 h before being incubated with or without 50 μM etoposide (Sigma, #E1383) for 24 h. Macrophages and A549 cells were co-cultured in CO₂ independent medium supplemented with 0.5 mM L-glutamine and 2.5 g/l of D-glucose for 24 h before being incubated with or without 50 μM etoposide for 16 h. In the monoculture experiments, 0.8 x 10⁶ THP-1 monocytes were differentiated and polarized in 6 well plates. Next, they were incubated in CO₂ independent medium supplemented with 0.5 mM L-glutamine (Sigma, # G3126) and 3.75 g/l of D-glucose (Sigma, #50-99-7) for 16 h before being incubated with or without 50 μM etoposide (Sigma, #E1383) for 24 h.