Phagocytic assay

BMDMs (5 x 10⁵ U.mL^-1) were cultured in 24 well plates containing 13.0 mm diameter glass coverslips and incubated with 200 μL of DMEN F-12 medium for 2 h for adherence at 37ºC/ 5% CO₂. Adherent macrophages were infected with promastigote forms (10:1) for 2h. After infection, free promastigotes were removed by washing with PBS, and the infected cells were treated with A, B, C and D fungal extracts (5.0 mg.mL^-1) and incubated for 24 h at 37ºC and 5% CO₂. DMEM F-12 medium was used as a control. L. amazonensis-infected BMDMs were stained with Giemsa and 20 fields analyzed using a light microscope (magnification of 1000x, OLYMPUS, model CX31RBSFA) to determine the phagocytic index by % of infection and the number of parasites/macrophage.