Influence of the culture medium on the production of nitric oxide and expression of inducible nitric oxide synthase by activated macrophages in vitro

• Activation macrophages phenotypes were different in DMEM and F-12 containing FBS.
• NO production and iNOS expression by LPS and IFN-γ were remarkably different.
• O2--generating activity, and production TNF-α and IL-1β were also different.
• The choice of culture medium is important for estimation of macrophage activation.

Macrophages play an important role in immune and inflammatory responses, and have been extensively studied in vitro using culture media such as RPMI1640 medium, Dulbecco's modified Eagle medium (DMEM), and Ham's F-12 medium (F-12). We found that the activation phenotypes of a murine macrophage-like cell line, J774.1/JA-4, were obviously different in two distinct culture media (F-12 and DMEM), both of which were supplemented with 10% of the same fetal bovine serum (FBS). Among these phenotypes, nitric oxide (NO) production as well as inducible NO synthase (iNOS) expression, induced by lipopolysaccharide (LPS) and interferon-γ (IFN-γ), were remarkably different. iNOS expression was higher in the macrophages cultured in DMEM than in F-12 for 20 h, while no significant differences were shown in NO production between in F-12 and DMEM. It might be the reason why DMEM have reduced NO production by the induced iNOS. Besides, O2−-generating activity, and production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the activated macrophages were also different between the cultures in F-12 and DMEM. These results suggest that F-12 and DMEM contain certain components responsible for modification of macrophage activation processes and/or macrophage functions. Our present results provide evidence that the choice of culture medium is important in the study and analysis of macrophage activation.