Lab tek

Differentiated BMDMs were removed from the substrate by pipetting of ice-cold PBS, seeded in 8-well culture chamber slides (Lab-Tek; BD Biosciences) at a density of 5 × 10⁴ cells per well in DMEM medium, and allowed to adhere overnight at 37 °C in a 5% CO₂ incubator. In order to perform the infection assay, L. infantum and L. amazonensis promastigotes as well as metacyclic L. major promastigotes isolated by the peanut agglutinin method [95 (link)] were used to generate infection at a macrophage/parasite ratio of 1/20. The plates were incubated for 24 h under the same conditions to promote promastigote phagocytosis. The wells were then washed with medium to remove the extracellular promastigotes and plates were incubated with fresh medium supplemented with increasing concentrations of compounds. After 48 h, cells were washed with PBS, fixed with ice-cold methanol for 5 min, and stained with Giemsa stain. To determine parasite burden, the number of amastigotes per infected macrophages was counted under a light microscope. The mean number of amastigotes per infected macrophage was determined by dividing the total number of amastigotes counted by the number of infected macrophages.

BALB/c mouse bone marrow monocytes were differentiated to macrophages using the fibroblast cell line L929 supernatant. Bone marrow-derived murine macrophages were then seeded in 8-well culture chamber slides (Lab-Tek; BD Bioscience, East Rutherford, NJ, USA) at a quantity of 50 × 10³ cells per well in supplemented DMEM medium, and allowed to adhere overnight at 37 °C in a 5% CO₂ incubator. For the infection assay, stationary-phase promastigotes were added at a macrophage/parasite ratio of 1/5 and incubated under the same conditions, allowing macrophage phagocytosis. Twenty-four hours later, wells were washed with preheated PBS to remove extracellular promastigotes, fixed with ice-cold methanol for 5 min, and stained with Giemsa stain. To determine the parasite burden, macrophages and amastigotes were counted under a light microscope until reaching 100 infected macrophages. The percentage of macrophage infection was determined by dividing the number of infected macrophages by the total number of macrophages counted. The number of amastigotes per infected macrophage was calculated by dividing the total number of amastigotes counted by the number of infected macrophages. The experiment was performed in quadruplicate.