Tcrβ fitc

Briefly, the small intestine was placed in Hank’s Balanced Salt Solution (HBSS) buffer supplemented with 10% fetal bovine serum. Peyers patches were excised and tissue was placed in digestion media containing 1 mM DTT, 1 mM EDTA in calcium/magnesium-free HBSS supplemented with 2% fetal calf serum and subsequently treated with Collagenase IV/Dnase digestion mix (0.5 mg/ml of collagenase IV and 200 μg/ml of Dnase). Lymphocytes were enriched using a 44/67% discontinuous Percoll (GE Lifesciences, Pittsburgh PA) gradient. Splenic lymphocytes were treated with ammonium chloride and washed. Cells were stimulated with phorbol 12-myristate 13-acetate and Ionomycin for 4 h at 37 °C in the presence of brefeldin A (GolgiPlug; BD Bioscience, San Jose, CA). Following stimulation, cells were stained with LIVE/DEAD Fixable Aqua (ThermoFisher Scientific, Waltham, MA), and the following antibody/fluorophore combinations APCC7-CD45, TCRβ-FITC, CD4-V500 (BD Bioscience), CD8-BV650, Foxp3-PECy7, IL17-PE, IFNγ-FITC (eBioscience, San Diego, CA), and fixed with fix/perm (eBioscience), were used according to the manufacturer’s instructions. Cells were acquired on an LSRII flow cytometer (BD Bioscience) and analyzed with FlowJo software (Tree Star, Ashland, OR); 100,000 events or greater were collected for each sample. Samples with yields less than 10,000 viable events were excluded from analysis.

Thymocytes were stained with the following antibodies: Ter119-biotin, GR-1-biotin, B220-biotin, NK1.1-biotin, Streptavidin-PE-Cy7, CD3-APC, TCRβ-FITC (BD Biosciences, Franklin Lakes, NJ, USA), CD11b-biotin, CD8-PerCP (Biolegend, San Diego, CA, USA), CD45.2-APC-eFluor780, CD4-BV650 (eBioscience, Santa Clara, CA, USA). The biotinylated antibodies were used to exclude lineages other than the T cell lineages. Cells were stained in FACS buffer (PBS, 2% BSA, 0.1% sodium azide) and subsequently measured by flow cytometry.