Macrophage Phagocytosis of Paracoccidioides

Thioglycollate-induced peritoneal macrophages were isolated by adherence (2 h at 37°C in 5% CO₂) to plastic-bottom tissue-culture plates (1×10⁶ cells/well in 24 well plates). Macrophages were washed to remove nonadherent cells and cultivated overnight with fresh complete medium (DMEM, Dulbecco's Modified Eagle's Medium, Sigma, containing 10% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin) in the presence or absence of recombinant IFN-γ (20 ng/ml in culture medium, BD-Pharmingen) and/or 1MT (1-methyl-d,l-tryptophan, Sigma-Aldrich). 1MT was used in the concentration of 1 mM that was previously shown to efficiently inhibit the IDO activity of macrophages [6] (link). Macrophage cultures were infected or not with P.brasiliensis yeasts in a macrophage∶yeast ratio of 25∶1 and cocultivated for 4 h. This ratio was previously determined and was shown to be non-deleterious to macrophage cultures and adequate for killing assays [38] (link). The monolayers were then washed to remove nonadherent cells and incubated for an additional 48 h period in the presence or absence of IFN-γ (20 ng/ml in culture medium, BD Biosciences) and/or 1MT.