ca/peptides).8,10 Fluorescein-conjugated killed Staphylococcus aureus was purchased from Molecular Probes (Karlsruhe, Germany). The E. coli strain JM109 was obtained from Promega (Mannheim, Germany). Cell culture reagents were purchased from BioWhittaker (Aachen,
Germany), PAA Laboratories (Coelbe, Germany) and Gibco-Life Technologies (Karlsruhe, Germany). Cell-permeable inhibitors of intracellular signalling molecules [SB203580, rottlerin, LY 294002 and janus kinase (JAK) inhibitor I pyridone 6] were purchased from Calbiochem (Nottingham, UK). Buffy-coats with blood cells for in vitro experiments buy BYL719 with human neutrophils and monocytes were obtained from healthy adult volunteers via the German Red Cross (Deutsches Alectinib manufacturer Rotes Kreuz, Münster, Germany). Neutrophils were isolated by Biocoll (Biochrom, Berlin, Germany) density gradient centrifugation followed by a hypotonic shock procedure.10,16 Peripheral blood monocytes were isolated by leukapheresis as previously described.17 Isolated human monocytes were cultivated in Teflon bags in McCoy’s medium (Biochrom) supplemented with 15% fetal calf serum (FCS), 2 mm l-glutamine and 1% non-essential amino acids. Monocytes were allowed to rest for 24 hr before stimulation. Isolated neutrophils were cultured in RPMI-1640 medium supplemented with 0·9% FCS, 2 mm l-glutamine and 1% non-essential amino acids and allowed to recover
for 2 hr before stimulation. The following concentrations of reagents were used for stimulation during experiments: LPS 100 ng/ml; IFN-γ 10 or 100 ng/ml; PAR2-cAP 1 × 10−4 m. The corresponding reverse peptide with the reverse-sequence (PAR2-cRP) was used at a concentration selleck kinase inhibitor of 1 × 10−4 m and served as a negative control. Bacterial killing
assay using E. coli (strain JM109) was performed as described previously18,19 with modifications. In brief, E. coli bacteria were cultured into Luria broth medium overnight at 37°. Isolated uninfected human neutrophils were pre-stimulated with 10−4 m PAR2-cAP and/or IFN-γ (100 ng/ml) for 2 hr (37°, 5% CO2). Unstimulated neutrophils were used as control samples. After 2 hr incubation of neutrophils with stimuli, the cell culture medium (RPMI-1640 with 0·9% FCS, 2 mm l-glutamine and 1% non-essential amino acids) with stimuli was removed and cells were washed. Human neutrophils (2 × 106 cells) were resuspended in 200 μl RPMI-1640 containing 2 mm l-glutamine, 1% non-essential amino acids, 0·2% BSA, 0·01% CaCl2 and 0·01% MgCl2 (this medium was designated the ‘assay medium’). Collected and washed bacteria (40 × 106 cells) were opsonized for 15 min at 37°. For opsonization, bacteria were incubated in the assay medium containing 5% human serum from the same donor from whom the neutrophils were obtained. After opsonization, bacteria were washed. Neutrophils and opsonized bacteria were co-incubated in assay medium in the absence (for unstimulated control samples) or presence of stimuli (10−4 m PAR2-cAP and/or 100 ng/ml IFN-γ) for 1 hr at 37° on a shaker.