Mice (female, 6-week-old, variety BALB/c) were from Research Institute of Animal Production (Velaz, Prague, Czech Republic). The mice had free access to standard pelleted diet and tap water. The animal facilities comply with the European Convention for the Protection of Vertebrate Animals Used for Experimental LY294002 nmr and Other Purposes. The experimental protocol was approved by the Ethics Committee and by the Slovak State Veterinary Committee

of Animal Experimentation. Mice (40 mice per one conjugate) were subcutaneously (sc) primary immunized (1st dose) with conjugate without adjuvant (6 μg oligosaccharide per dose) and subsequently primary sc boosted (2nd dose) without adjuvant 2 weeks after primary injection. Two weeks after primary booster injection, mice were divided

into two groups and were secondary boosted by sc (3rd sc dose) or intraperitoneal (ip, 3rd ip dose) administration of the same conjugate dose without adjuvant. Sera samples were collected at day 14 following each injection. Mice (10 mice in group) three times sc injected with saline in the same time schedule were used as controls. Yeast strain C. albicans CCY 29-3-100 (serotype A) (Culture Collection of Yeasts, Institute of Chemistry, Slovak Academy of Sciences, Slovakia) was cultured on 7% malt extract agar buy Cobimetinib at 28 °C. After 48 h, static cultivation cells were harvested in saline, washed twice with PBS pH 7.4. Viability was specified by flow cytometry with propidium iodide negative staining >99%. Fixation of Candida cells was carried

out very by mixing with 60% ethanol (45:5 v/v) and incubating 15 min at 25 °C, washed twice with PBS and adjusted to 5 × 106 cells/ml with PBS. Ethanol-killed Candida cells were used as control sample in flow cytometric analysis for electronic gates set-up. Levels of induced anti-mannan sera immunoglobulins (IgG, IgM and IgA) were determined by ELISA test, using C. albicans serotype A, C. albicans serotype B or C. guilliermondii mannan in coating step [18]. Antibodies levels were detected at serum dilution 1:100 (n = 10 mice from each group). For the exact expression of IgG, IgM and IgA levels, quantification (in ng/ml) using appropriate calibration curve based on reference mouse serum (Mouse Reference Serum; Bethyl Laboratories, Inc., Montgomery, TX, USA) was done. Statistic analysis was performed using one-way ANOVA test. All data were expressed as mean ± SD. P-values <0.05 were considered statistically significant. Induced C. albicans CCY 29-3-100 (serotype A) whole cell–specific sera immunoglobulin levels (IgG, IgM and IgA, n = 10 mice from each group) were determined by whole cell ELISA test, using C. albicans serotype A cells as yeast and hyphal morphological forms in plate-coating step. The concentrations of coated substances and C.