The cells were then washed 3 times with PBS, re-plated with complete medium, and allowed to rest overnight. For the coculture of BMDCs with syngeneic splenocytes in a mixed leukocyte reaction, the spleens were harvested, crushed, and filtered through 100 ��m filters and the red blood cells were lysed with ammonium-chloride-potassium (ACK) lysing solution. selleck Tipifarnib The mixed leukocyte reaction was performed with a splenocyte-to-BMDC ratio of 10 to 1. Supernatants were collected for cytokine analysis at 72 h. Animal studies C57BL/6 and TLR2KO mice were orally gavaged with H. pylori SS1 (3 times over 1 week). After 2 months, the mice were analyzed. The stomach was cut along the greater curvature and removed. Strips (2 mm) composed of fundus and antrum were embedded in Tissue-Tek optimum cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA) and immersed in liquid nitrogen.

Paraffin sections were also prepared for H&E staining. Meanwhile, splenocytes from C57BL/6 and TLR2KO mice were cocultured for 18 h with BMDCs infected with H. pylori. The splenocyte-to-BMDC ratio was 10 to 1. After 72 h, splenocytes were collected and the percentages of IFN-��, IL-17A, and Foxp3 were measured by flow cytometry (i.e., FACS, fluorescence-activated cell sorting). Measurement of cytokine production by BMDCs and splenocytes The supernatant from the coculture of BMDCs and splenocytes was collected and the concentrations of IL-12p70, IL-23p19, IL-6, TNF-��, IFN-��, IL-10, and IL-17A were measured using Quantikine ELISA Kits (eBioscience, San Diego, CA, or BD Biosciences, San Jose, CA).

Determination of the expression of intracellular markers on splenocytes and surface markers on BMDCs by flow cytometry Splenocytes were labelled with fluorescence-conjugated antibodies to Foxp3, IFN-��, and IL-17A (eBioscience). Stimulated BMDCs were dual-labelled with fluorescence-conjugated antibodies to CD11c and TLR2 or TLR4. For cytokine profiles, the cells were stimulated, stained, and analyzed, as previously published [20], with a Coulter XL Flow Cytometer (Beckman Coulter, Miami, FL). Histological analysis After H. pylori infection of 2 months duration, the stomachs of WT and TLR2KO mice were removed and measured. Two adjacent full-thickness longitudinal strips were removed from the greater curvature and fixed in formalin for histological analysis.

The specimens were scored separately for the presence Cilengitide or absence of each of the following histologic criteria: neutrophil infiltration (polymorphonuclear neutrophils), gastritis, and epithelial metaplasia. The results were reported as the percentage of fields affected each slide [21]. Immunohistochemistry Paraffin sections (8��m) were de-waxed in xylene twice, incubated in 100%, 95%, and 70% ethanol for 1 minute each, and hydrated in water for 5 minutes.