Syk inhibitor piceatannol

For investigation of the role of selected receptors or signal mediators in netosis, neutrophils were preincubated with specific antibodies or inhibitors prior to stimulation with C. albicans factors. Neutrophils (1 × 10⁶) were preincubated for 30 min at 37°C in RPMI-1640 medium with 1 μg/ml of blocking antibodies directed against TLR2, TLR4, Dectin-1, CD14 (Invivogen, Toulouse, France), CD11a, CD11b, CD16, CD18 (BioLegend, San Diego, CA, USA) or isotype control antibody—IgG (Abcam). For the analysis of selected signaling pathways, neutrophils (1 × 10⁶) were preincubated for 30 min at 37°C in RPMI-1640 medium with 30 μM of Syk inhibitor (piceatannol; Sigma-Aldrich), 10 μM of Src inhibitor (PP2; Calbiochem, Darmstadt, Germany), 25 μM of PI3K inhibitor (LY29004; Calbiochem), or 10 μM of ERK inhibitor (UO126; Cell Signaling Technology, Beverly, MA). To inhibit ROS-dependent netosis pathways, neutrophils were preincubated for 30 min at 37°C in RPMI-1640 medium with 10 μM DPI (Sigma-Aldrich), an NADPH oxidase inhibitor. Phagocytosis of neutrophils was blocked by pretreatment of the cells with 5 μM of cytochalasin D (CytD, Sigma-Aldrich) for 15 min at 37°C. After removal of the blocking agent by cell washing, neutrophils were treated with the indicated stimulants. Untreated neutrophils were used as controls.