In (BSA) in PBS for 20 minutes at room temperature. Afterwards, the

In (BSA) in PBS for 20 Title Loaded From File minutes at room temperature. Afterwards, the sections were incubated overnight with anti-CD3 antibodies (Dakocytomation) in 1 BSA/PBS for 45 minutes. After thoroughly washing with PBS, 1317923 1480666 the slides were incubated with biotinylated goat-antirabbit antibodies (Dakocytomation) and streptavidinhorseradish peroxidase (Vectastain Elite ABC, Vector Laboratories, Burlingame, CA USA) for 45 minutes at room temeperature. Specific binding was detected by incubating the sections with 0.05 diaminobenzidine-tetrahydrochloride (Sigma-Aldrich, St. Louis, MO USA)/0.015 H2O2/0.01 M TrisHCL, pH 7.6 for 10 minutes resulting in a brown staining product. Sections were counterstained with Mayers’ haematoxylin (Merck, Darmstadt, Germany), dehydrated and mounted. Slides without primary antibody incubations were included as negative controls. Photomicrographs were taken with an Olympus BX50 microscope equipped with a Leica DFC 320 digital camera. Contrast of the H E stained sections was improved and magnification bars were added using Adobe Photoshop CS5.Detection of memory T cellsSingle cell suspensions were first washed with ice cold 1 BSA/PBS. To prevent background staining, cells were first incubated with unlabeled anti-CD16/32 (eBioscience, San Diego, CA USA) for 15 minutes on ice. Cell samples were then stained with antibodies specific for mouse CD3, CD4, CD62L, and CD44 (eBioscience) in the dark, on ice for 30 minutes. After washing with 1 BSA/PBS, the samples were measured with a BD FACSCantoTM II (BD Biosciences, Franklin Lakes, NJ USA). Analysis of the flow cytometry data was performed using BD FACSDiva software (BD Biosciences).Intracellular cytokine stainingSingle cell suspensions of mesenteric lymph nodes and spleens were cultured in Roswell Park Memorial Institute medium (RPMI, Life Technologies, Paisley, Scotland) supplemented with 1 unit/ml penicillin, 1 /ml streptomycin, 50 -mercaptoethanol and 5 FCS in 96 well round-bottom plates at a concentration of 105 cells per well. Intracellular cytokine staining was performed on cells after 24 hours stimulation with purified anti-CD3 (eBioscience) at 2 /ml in the medium. Cells were then stained with antibodies for CD4, IFN and IL-17A (all antibodies from eBioscience) using the Foxp3 intracellular staining kit (eBioscience). To prevent background staining, cells were first incubated with unlabeled anti-CD16/32 (eBioscience) for 15 minutes on ice as suggested in the manufacturer’s protocol. Fixed samples were kept at 4 until reading with the flow cytometer. Samples were measured using a BD FACSCanto II flow cytometer. Analysis of the FACS data was performed using BD FACSDiva software (BD Biosciences).Serum amyloid A measurementSerum was Title Loaded From File collected on day 14 via heart puncture and tested for serum amyloid A (SAA). The SAA ELISA was obtained from Invitrogen (Paisley, UK) and was used according the manufacturer’s directions. The results were read using the Bio-Rad (Hercules, CA USA) iMarkTM microplate reader and analyzed using Microplate Manager 6.1 software (Bio-Rad).Isolation of lymphoid organs and colon cellsMesenteric lymph nodes (mLNs) and spleens were excised from mice and kept on ice in PBS. To make single cell suspensions, the organs were crushed and the slurry was filtered using 70 filters to collect the single cells. The spleens were further purified by destroying the red blood cells by isotonic shock. Cell counts in mLN and spleen were determined using a Coulter Cou.In (BSA) in PBS for 20 minutes at room temperature. Afterwards, the sections were incubated overnight with anti-CD3 antibodies (Dakocytomation) in 1 BSA/PBS for 45 minutes. After thoroughly washing with PBS, 1317923 1480666 the slides were incubated with biotinylated goat-antirabbit antibodies (Dakocytomation) and streptavidinhorseradish peroxidase (Vectastain Elite ABC, Vector Laboratories, Burlingame, CA USA) for 45 minutes at room temeperature. Specific binding was detected by incubating the sections with 0.05 diaminobenzidine-tetrahydrochloride (Sigma-Aldrich, St. Louis, MO USA)/0.015 H2O2/0.01 M TrisHCL, pH 7.6 for 10 minutes resulting in a brown staining product. Sections were counterstained with Mayers’ haematoxylin (Merck, Darmstadt, Germany), dehydrated and mounted. Slides without primary antibody incubations were included as negative controls. Photomicrographs were taken with an Olympus BX50 microscope equipped with a Leica DFC 320 digital camera. Contrast of the H E stained sections was improved and magnification bars were added using Adobe Photoshop CS5.Detection of memory T cellsSingle cell suspensions were first washed with ice cold 1 BSA/PBS. To prevent background staining, cells were first incubated with unlabeled anti-CD16/32 (eBioscience, San Diego, CA USA) for 15 minutes on ice. Cell samples were then stained with antibodies specific for mouse CD3, CD4, CD62L, and CD44 (eBioscience) in the dark, on ice for 30 minutes. After washing with 1 BSA/PBS, the samples were measured with a BD FACSCantoTM II (BD Biosciences, Franklin Lakes, NJ USA). Analysis of the flow cytometry data was performed using BD FACSDiva software (BD Biosciences).Intracellular cytokine stainingSingle cell suspensions of mesenteric lymph nodes and spleens were cultured in Roswell Park Memorial Institute medium (RPMI, Life Technologies, Paisley, Scotland) supplemented with 1 unit/ml penicillin, 1 /ml streptomycin, 50 -mercaptoethanol and 5 FCS in 96 well round-bottom plates at a concentration of 105 cells per well. Intracellular cytokine staining was performed on cells after 24 hours stimulation with purified anti-CD3 (eBioscience) at 2 /ml in the medium. Cells were then stained with antibodies for CD4, IFN and IL-17A (all antibodies from eBioscience) using the Foxp3 intracellular staining kit (eBioscience). To prevent background staining, cells were first incubated with unlabeled anti-CD16/32 (eBioscience) for 15 minutes on ice as suggested in the manufacturer’s protocol. Fixed samples were kept at 4 until reading with the flow cytometer. Samples were measured using a BD FACSCanto II flow cytometer. Analysis of the FACS data was performed using BD FACSDiva software (BD Biosciences).Serum amyloid A measurementSerum was collected on day 14 via heart puncture and tested for serum amyloid A (SAA). The SAA ELISA was obtained from Invitrogen (Paisley, UK) and was used according the manufacturer’s directions. The results were read using the Bio-Rad (Hercules, CA USA) iMarkTM microplate reader and analyzed using Microplate Manager 6.1 software (Bio-Rad).Isolation of lymphoid organs and colon cellsMesenteric lymph nodes (mLNs) and spleens were excised from mice and kept on ice in PBS. To make single cell suspensions, the organs were crushed and the slurry was filtered using 70 filters to collect the single cells. The spleens were further purified by destroying the red blood cells by isotonic shock. Cell counts in mLN and spleen were determined using a Coulter Cou.