Macrophages phagocytic and microbicidal activity assays

Macrophages were obtained from the peritoneal cavity of BALB/c mice 72 h after an intraperitoneal injection of 1 mL of 3% sterile sodium thioglycollate medium (Sigma-Aldrich), according to the described protocol (Freitas et al., 2016). Peritoneal macrophages (1 × 10⁶) were stimulated or not with 10 μg of rPb18_Dld and maintained in a 5% CO₂ humidified atmosphere incubator at 37°C. After 24 h, macrophages were infected with 3 × 10⁵ yeast cells of P. brasiliensis and incubated for 4 h at 37°C. The cultures were washed with RPMI-incomplete medium. To evaluate the phagocytic activity, 1 mL of distilled water was added to the microplate wells to promote macrophage lysis. The samples were serially diluted and cultured for 10 days at 37°C in solid BHI medium supplemented with 1% glucose to determine the presence of colony forming units (CFU) of viable yeasts. To analyze microbicidal activity, the washed cultures were additionally incubated for 48 h. Macrophage lysis and yeast cultures were carried out as described above. The number of CFU were counted and expressed as CFU/well.