Determination of cytokine mRNA expression in intestinal tissue The samples were processed www.selleckchem.com/products/MDV3100.html as described above (see Determination of ZO-1 mRNA expression in intestinal tissue). Gene expression assays for IL-10, IL-6, TNF-�� and ��-actin were all purchased by Applied Biosystems, Foster City, CA, USA. Determination of specific antibodies Sera and small intestine washings were collected for specific antibody evaluation. Gut washings were obtained by flushing the content of isolated small intestine with 2 ml of sterile PBS containing a mixture of proteinase inhibitors (Sigma-Aldrich). The samples were then vortexed and centrifuged at 4��C, and the supernatant was collected and stored at ?80��C until analysis.

Indirect ELISA, optimized in our laboratory as previously described [11], was used to assess the specific antibody response against Lc in serum (IgG, IgM, and IgA) and gut washings (secretory IgA; SIgA). Briefly, Nunc MaxiSorp 96-well plates (Thermo Fisher Scientific Inc., Rochester, NY, USA) were coated overnight with Lc (100 ��l/well at 10 mg/l in PBS) and blocked with 1% BSA (Sigma-Aldrich) in PBS. Serum and gut washing samples diluted 150 and 110 in 1% BSA, respectively, were added and incubated for 2 hours. As control sera normal reference serum purchased from Bethyl Laboratories (TX, USA) and hyperimmune serum prepared by four subcutaneous injections of Lc in incomplete Freund’s adjuvant within 14 days intervals (50 ��g of Lc in the each dose) were used. After washings (three times with PBS containing 0.

05% Tween 20 (Sigma-Aldrich)), secondary antibodies (50 ��l/well) were added and incubated for 1 hour at room temperature. Antibody combinations were used as follows: 1) rabbit anti-mouse SIgA (Uscn Life Science Inc., China) and horseradish peroxidase (HRP)-labeled anti-rabbit IgG (Cell Signaling Technology Inc., Danvers, MA, USA); 2) biotinylated anti-mouse IgA (Sigma-Aldrich) and streptavidin-HRP (R&D Systems Inc.); 3) HRP-labeled anti-mouse IgG; 4) HRP-labeled anti-mouse IgM (both The Binding Site Ltd, Birmingham, UK). All reagents were diluted in 1% BSA in PBS except anti-IgA antibody that was diluted in 1% BSA with 5% fetal bovine serum (BioClot GmbH, Aidenbach, Germany). The plates were developed with 3,3��,5,5��-tetramethylbenzidine (Sigma-Aldrich) and the optical density (OD) was measured at 450 nm.

The OD of the background (1% BSA) was subtracted and resulting adjusted ODs of the treated groups were compared with those of PBS-treated groups. Flow cytometry Single-cell suspensions of spleens, MLNs and PPs were prepared and stained for Tregs using FoxP3 Staining Set (eBioscience, GSK-3 San Diego, CA, USA) with fluorochrome-labeled anti-mouse mAbs: CD4-Qdot? 605 (Invitrogen, Carlsbad, CA, USA), CD8-BD Horizon? V500 (BD Biosciences, San Jose, CA, USA), CD3-FITC and FoxP3-Phycoerythrin (both from eBioscience) according to the manufacturer’s recommendation. RAW 264.