4 and incubation temperature of 23 °C. Parasites used for in vitro tests were removed from culture at

the stationary phase, at the seventh passage. Blood samples from the five healthy dogs, 20 mL each, were collected in heparinized tubes intended for obtaining PBMCs. The whole blood volume collected was placed in a mixture of Ficoll–Hypaque (Sigma Chemical Co., USA, density: 1.119 g/mL) and Ficoll–Hypaque (Sigma Chemical Co., USA, density: 1.077 g/mL) at a 1:3 ratio (Ficoll/blood) in sterile polystyrene conical bottom tubes (Falcon™, Corning®, USA). All samples Epigenetics inhibitor were centrifuged at 700 × g for 80 min at 22 °C. The ring of mononuclear cells collected at the Ficoll–Hypaque interface was transferred to another tube with 40 mL of Falcon sterile 1× PBS containing 10% FBS. This tube was centrifuged two times at 400 × g for 10 min at 4 °C. After the supernatant was discarded, the cells were resuspended in 1 mL of cell culture medium Selumetinib supplier RPMI 1640.

Cells were counted in a Neubauer hemocytometer chamber to determine the numbers of monocytes or lymphocytes per milliliter. After counting cells in a Neubauer chamber, we calculated the percentage of monocytes that were plated at 5 × 105 monocytes/well using 24-well plates (Thermo Fisher Scientific Inc., NUNC, USA), on circular coverslips (15 mm; Glasscyto, BRA). Cultures were established using RPMI supplemented with 20% fetal calf serum (FCS) and 20% macrophage colony-stimulating factor (M-CSF) medium and incubated at 37 °C/5% CO2. The M-CSF was obtained from supernatant of cultures of L929 immortalized cells. After 24 h, the wells were gently washed to removed nonadherent cells, which were then transferred to new 48-well plates (Thermo Fisher Scientific Inc., NUNC, USA) and grown for 4 days in RPMI/20% FCS, at which time purification of CD4+ and CD8+ T lymphocytes was undertaken. To determine the timing of monocyte differentiation into macrophages with high phagocytic and microbicidal activity, distinct conditions were analyzed in duplicate. Monocytes differentiating into macrophages were

evaluated PD184352 (CI-1040) from 2 to 5 days of culture. In all conditions, the cells were infected with 5 × 106 of L. chagasi promastigotes in the stationary phase, using a 10:1 ratio (10 parasites per macrophage). Each well was washed gently 3 h after infection and cultures were maintained to assess microbicidal activity 24, 48, 72, and 96 h postinfection. For the rate of parasitic infection, we counted the numbers of amastigotes in 200 macrophages. Thus, the total number of amastigotes was divided by the total number of infected macrophages in order to obtain the average number of amastigotes per macrophage. NAG analysis served as an indicator of cellular activation levels after in vitro infection for various differentiation times of monocytes and macrophages. Supernatant from macrophages cultured for 2–5 days was submitted to in vitro infection with L.