Mice that received pretreatment with Con-A or PBS were infected with C. albicans and sacrificed after 30 min, 2, 6, 18, and 24 h, and their peritoneal exudates were collected with 2 mL RPMI 1640 medium (Sigma-Aldrich) pH 7.0 with 14 μg gentamicin. For cytokine analysis, the supernatants were

collected by centrifugation (800 g × 4 min at 4 °C). To evaluate the population of collected cells from the peritoneal exudate cells, the pellet MG-132 supplier was resuspended in 1 mL RPMI medium pH 7.0 supplemented with 5% inactivated fetal calf serum (FCS) plus 7 μg gentamicin. The peritoneal exudate cells collected following infection were adhered to coverslips (0.2 mL per coverslip) for 30 min at 37 °C. The following tests were applied to the cells: (1) staining by May-Grunwald-Giemsa (Merck, Darmstadt, Germany) and analysis by light microscopy to evaluate the populations of macrophages and neutrophils, percentage EPZ-6438 supplier of phagocytosing cells by counting 20 fields; (2) staining with propidium iodide plus 6-carboxyfluorescein

diacetate (6-CFDA; Sigma-Aldrich) to evaluate the presence of necrotic and viable cells, as described by Gasparoto et al. (2004). Analysis of fluorescence labeling was performed in a fluorescent microscope (Zeiss) and photographed (blue-violet irradiation: BG-38 and BG-12 excitation filters and 530-barrier filter), and (3) the cells were fixed in 2.5% glutaraldehyde (Merck) buffered with phosphate 0.1 M for 2 h at room temperature

followed by postfixation in osmium tetroxide 1% for 1 h. Following dehydration in ethanol, the samples were dried by the critical-point method, coated with a thin layer of gold and examined in a scanning microscopy (Shimadzu 550 SS) after 2 h of phagocytic assays in vitro. The mice received intraperitoneally Con-A 250 μg per 250 μL PBS or PBS alone 72 h before phagocytic assays. To study Dectin-1, monolayers of peritoneal macrophages 4 × 105 were preincubated with laminarin (Sigma-Aldrich) 100 μg mL−1 PBS for 30 min at 37 °C (Gantner et al., 2005). To study mannose receptors, macrophages were preincubated with mannan (Sigma-Aldrich; 100 μg mL−1 PBS; Gaziri et al., 1999). Fresh laminarin or mannan were Y-27632 2HCl added to C. albicans 2 × 106 and coincubated with macrophages at 37 °C for 30 min. A total of 200 phagocytes were analyzed for each preparation, and the percentage of phagocytes that phagocytosed Candida was determined following staining of the cells with May-Grumwald-Giemsa (Merck). Supernatants were collected after centrifugation of peritoneal exudates obtained from each mouse from all the experimental groups and submitted to capture ELISA (eBioscience, San Diego, CA) to determine the concentrations of IL-17, TGF-β, IL-1β, IL-6, IL-12, IFN-γ and TNF-α. Cytokines assays were performed according to the manufacturer’s instructions. The differences between groups were analyzed by the Student’s t-test. P < 0.