Data (an average of 10,000 events per sample) were analysed with

Data (an average of 10,000 events per sample) were analysed with the PD98059 solubility dmso cell quest Software (Cell Quest Software, San Jose, CA, USA). Evaluation of fungicidal activity.  After Pb18 challenge, neutrophil–fungus cocultures were harvested by aspiration with sterile distilled water to lyse neutrophils. Washing of each well resulted in a final volume of 2.0 ml, and 0.1 ml was plated on supplemented brain–heart infusion agar medium (Difco Laboratories, Detroit, MI, USA) plates containing 0.5% of gentamicin, 4% horse normal serum and 5%P. brasiliensis strain

192 culture filtrate (vol/vol), the latter being the source of growth-promoting factor. Inoculated plates, in triplicate of each culture, were incubated at 35 °C in sealed plastic bags to prevent drying. After 10 days, the number of colony forming units (CFU) per plate was counted. The inoculum used for the challenge was also plated according to the same conditions. The plates containing the material obtained from the neutrophil–fungus cocultures were considered as experimental plates, and those plated with the inoculum alone and counted at time zero were used as control plates. Fungicidal activity percentage was determined by the following formula: % Fungicidal Activity = [1−(mean CFU recovered on experimental plates/mean CFU recovered on control plates)] × 100. Evaluation

of Compound Library H2O2 release.  The release of H2O2 by neutrophils was measured by the horseradish peroxidase–phenol red oxidation method [32]. For this assay, neutrophil cultures were Adenosine triphosphate challenged with Pb18 suspension diluted in phenol red buffer containing 50 μg/ml of horseradish peroxidase (type II, Sigma-Aldrich) plus 10% fresh human AB serum and further incubation for 1 h in 5% CO2 at 37 °C in humidified chamber. The reaction was stopped by addition of 10 μl of 1 N NaOH, and the absorbance at 620 nm was determined with a micro-ELISA reader (MD 5000; Dynatech Laboratories, Inc., Chantilly, VA, USA). All measurements were repeated four times, and the absorbance was converted

into nanomoles of a standard curve of H2O2. Measurement of cytokines.  After Pb challenge, neutrophil culture supernatants were separated from cell debris by centrifugation at 1000 g for 15 min and stored at −70 °C. TNF-α, IL-6, IL-8 and IL-10 concentrations were measured by capture ELISA using Kit DuoSet (R&D Systems). ELISA was performed according to the manufacturer’s protocol. Cytokine concentrations were determined with reference to a standard curve for serial twofold dilutions of recombinant cytokines. Absorbance values were measured at 492 nm using a micro-ELISA reader (MD 5000; Dynatech Laboratories). Statistical analysis.  Data were analysed statistically using the instat software (Graph Pad, San Diego, CA, USA). The results were compared by variance analysis (anova) followed by Tukey’s test, with the level of significance set at P < 0.05.

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