Macrophage Polarization Protocols for HBCs and THP-1 Cells

Treatment of HBCs was performed in complete MaM medium, treatment of THP-1 macrophages in complete RPMI-1640 and performed in T25 nunclone flasks (Nunc, Waltham, MA, USA) at a cell density of 1 × 10⁶ cells/mL. For induction of polarization towards M1 phenotype, lipopolysaccharide (LPS, Sigma, St. Louis, MO, USA) and Interferon gamma (INF-γ, Sigma, St. Louis, MO, USA) were added to a final concentration of 50 and 20 ng/mL, respectively. For induction of the M2 phenotype, interleukin 4 and 13 (IL-4, IL-13, both Sigma, St. Louis, MO, USA) were added to a final concentration of 20 ng/mL each. Both cell types were cultivated for 72 h after addition of stimulating agents. Untreated controls were included in each individual experiment. THP-1 macrophages were harvested after 72 h treatment and used for subsequent analysis. HBCs received a second treatment with stimulating substances and were cultivated for an additional 24 h before harvesting and proceeding. Preliminary experiments on HBCs from our group and also others [56 (link)] have shown that the isolation protocol using extensive digestion steps can reduce presence of surface markers in FACS and that up to 72 h recovery period are needed before e.g., CD163 is fully present again. Therefore it was decided to offer HBCs more time in culture exposed to respective stimuli.