Rorγt bv421 q31 378

Lymph node single cell suspensions and small intestinal tissue digestion for the isolation of siLP cells were performed as described previously (35 (link)). Cultures were kept for 6 h with brefeldin A added after 1 h before surface and intracellular staining. Surface and intracellular markers were stained according to the manufacturer's instructions with the following antibodies obtained from ThermoFisher/eBioscience, if not stated otherwise: CD4-PerCP/-BV510/-A700 (RM4-5), Foxp3-FITC/-PerCP-Cy5.5 (FJK-16s), GATA-3-A660/-PE/-PE-eF610 (TWAJ), T-bet-PE/-PE-Cy7 (eBio4B10), RORγt-BV421 (Q31-378, BD biosciences), IL-10-APC (JES5-16E3), IL-4-PE/-PE-Cy7 (11B11), and IL-17A-PerCP-Cy5.5 (eBio17B7). Live/dead discrimination was performed using fixable viability dye eF780 (ThermoFisher/eBioscience). Unspecific binding was prevented by addition of 20 μg/ml FcgRII/III blocking antibody (2.4G2).

Lymphocytes were isolated from the female reproductive tract of mice 16 days post infection. The FRT was harvested into complete RPMI, diced, then incubated while stirring with collagenase IV (386mg/L MP Biomedicals) for 1 hour at 37°C. Cells were filtered (70μm cell strainer, Corning), then lymphocytes were isolated out of the supernatant using a Percoll gradient (GE Healthcare). Cells were stimulated with PMA (0.2 mM, Millipore Sigma) and Ionomycin (1ug/mL, Millipore Sigma) with Brefeldin A (71.4uM Millipore) for 3.5 hours at 37°C 5% CO₂. Cells were stained for viability with Zombie Yellow (BioLegend) and surface markers B220-APC-eF780 (RA3-6B2, eBioscience), CD11b-APC-eF780 (M1/70, eBioscience), CD11c-APC-eF780 (N418, eBioscience), F4/80-APC-eF780 (BMB, eBioscience), CD4-PE (RM4-4, eBioscience), CD4-eF450 (RM4-5, eBioscience), CD8-PerCPCy5.5 (2.43, Tonbo), CD44-APC (IM7, eBioscience) CD62L-PETexasRed (MEL-14, Invitrogen), followed by intracellular stains IFN-γ-BV785 (XMG1.2, BioLegend), IL-4-PE (11B11, eBioscience), T-bet-PECy7 (4B10, eBioscience), RORγt-BV421 (Q31-378, BD Biosciences), and IL-17A-FITC (17B7, eBioscience) using the Foxp3 Transcription Factor Staining Kit (eBioscience). Data was acquired on an LSRFortessa (BD) and analyzed using FlowJo (Tree Star, San Carlos, CA). Contour plots are shown with 5% outliers.