pylori (Pellicanòet al., 2007). To date, the effects of IFN-γ on H. pylori have never been studied. To explore the effects, we designed an experiment to determine IFN-γ binding to H. pylori, protein profiles of H. pylori exposed to IFN-γ and the CagA protein levels in IFN-γ-treated H. pylori and in AGS gastric epithelial cells infected by IFN-γ-treated Selleck Ceritinib H. pylori. The H. pylori strains used in

this study were standard strains 26695 and SS1; both were cagA- and vacA-positive strains. Helicobacter pylori strains were grown in Brucella broth medium supplemented with 10% fetal calf serum (FCS), at 37 °C, in a microaerobic environment (5% O2, 10% CO2 and 85% N2). After culture to an exponential phase of growth, each bacterium was incubated with IFN-γ (ClonGamma, China) of various concentrations Sunitinib mw (0.065, 0.65, 6.5 and 65 ng mL−1). At 1-h intervals, the OD600 nm value was measured, and cell morphologic features were observed. Then, the bacteria were diluted and cultured in Skirrow agar plates containing 5% (v/v) sheep blood for 72 h; colonies were counted to determine the growth rate of H. pylori in the medium supplemented with and without IFN-γ. Cultured H. pylori exposed to IFN-γ at different concentrations was harvested after 2 h and washed three times with

phosphate-buffered saline (PBS is standard solution). The bacteria were fixed in a mixture of acetone and ethanol (v/v=3/2). After being co-incubated with anti-human IFN-γ antibody (1 : 200 dilution, Zhongshan, China) for 45 min below at 37 °C, the bacteria were washed with PBS five times (10 s each time). Then, fluoresceinisothiocyanate-labeled antibody (1 : 50 dilution, Zhongshan) was used to detect the binding of IFN-γ for 45 min at 37 °C. Bacteria were washed with PBS five times (10 s each time), and then observed under a fluorescence microscope. Helicobacter pylori bacteria were exposed to IFN-γ (65 ng mL−1), harvested by centrifugation after 6 h and washed three times with sterilized ice-cold PBS, then resuspended in lysis buffer (8 M urea,

4% 3-[(3-chloramidopropyl) dimethylammonium]-1-propanesulfonate, 1% dithiothreitol, 4 mM Tris, 1% pharmalyte, pH 3–10, 10 μg mL−1 protease inhibitor, 10 μg mL−1 RNase, 10 μg mL−1 DNase) and sonicated at 120 W, 5-min pulse: 1 s on, 3 s off. The solution was centrifuged and protein was obtained. Protein concentrations were determined using the Bradford method. About 300 μg protein was added to 18-cm IPG strips (pH 3–10) and placed on an IPGphor instrument (Amersham Biosciences, UK). The strips were rehydrated to 80 kVh, and then equilibrated for 15 min in buffer [50 mM pH 8.8 Tris-HCl, 6 M urea, 30% glycerol, 2% sodium dodecyl sulfate (SDS), a trace of bromophenol blue] with 0.5% (w/v) dithiothreitol and 2% (w/v) iodoacetamide.