Rigf 1

The RAW 264.7 macrophage cell line (ATCC) and BALB/c and C57BL/6 peritoneal macrophages were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 0.5% bovine serum albumin (BSA, insulin-free, Sigma, USA). Cells (5 × 10⁵ or 2 × 10⁶) were dispensed onto round 13 mm² glass cover slips, which were placed in the wells of 24-well plates (Corning Costar, USA) and incubated for 30 minutes at 37°C in a humid atmosphere with 5% CO₂ to allow adhesion. Thereafter, the wells were washed twice with culture medium to remove nonadherent cells. Then, the amastigote or promastigote suspensions (eight parasites per cell) were dispensed into the wells, and infection was allowed to occur for 4 hours at 33°C in a humid atmosphere with 5% CO₂. After incubation, the noninternalized parasites were washed away. In one set of experiments, macrophages were treated with the Th1 cytokine IFN-γ (200 U/mL) or the Th2 cytokines IL-4 (2 ng/mL) and IL-13 (5 ng/mL), both separately and in combination. In other experiments, recombinant IGF-I (50 ng/mL; rIGF-I, R&D Systems, Minneapolis, MN, USA) was added. The culture was then maintained for 48 hours at 37°C in a humid atmosphere with 5% CO₂.

L. (V.) braziliensis (MHOM/BR/1975/M2903) promastigotes were maintained at 25°C, in Schneider's insect medium (Gibco, Thermo Fisher Scientific Inc, MA, USA) supplemented with 10% heat-inactivated fetal calf serum (Gibco, USA) and 200 IU of penicillin per mL, and 200 μg of streptomycin (Sigma Chemical Company, St. Louis, Mo, USA) per mL, and grown until stationary phase before using in Leishmania growth curve analysis. The stationary phase promastigotes were distributed in triplicate into 24-well plates (5 × 10⁵ parasites/well) in a final volume of 1.0 mL of Schneider's insect medium (Gibco, USA) supplemented with 2% FCS and antibiotics, with or without 50 ng/mL recombinant human IGF-I (rIGF-I, R&D Systems, USA) and maintained during the experimental period.