Il 17a fitc

Intracellular cytokine staining was conducted on day 3, after the T cells were cultured for 72 h. The cultured cells were stimulated with Cell Stimulation Cocktail (eBioscience) for 5 h. Brefeldin A 1000X (eBioscience) was added for the final 4.5 h. Cells were then fixed and permeabilized with Intracellular Fixation & Permeabilization Buffer (eBioscience) or Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and intracellular cytokines overnight at 4 °C. Cells were stained with LIVE/DEAD Fixable Blue Dead Cell Stain Kit for UV Excitation (Invitrogen) or Ghost 780 (Tonbo) prior to fixation. Cells were stained with CD4 (BUV395; BD or PE, PeCy5; eBioscience) and TCRB (PeCy7; eBioscience) for extracellular markers. Cells were stained with IL-17A (FITC; eBioscience), IFNγ (eFluor450; eBioscience), and/or FOXP3 (eFluor450, eFluor660; eBioscience). Stained cells were analyzed on Fortessa (BD) or Attune NxT (Invitrogen). Data was analyzed using FlowJo software (TreeStar). Flow plots show cytokine producing cells as percent cytokine producing cells of CD4⁺TCRβ⁺ Live cells.

Spleen and MLN mononuclear cells were isolated and single-cell suspensions were prepared and live cells counted under a microscope. For the spleen an extra cell lysing step was carried out to remove the red blood cells (BD Biosciences). For cytokine staining, the cells were stimulated with 20 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma) and 1 μg/ml ionomycin (Sigma) in Iscove’s complete medium for 2 h at 37°C in a 5% CO₂ atmosphere. After 2 h, 10 μg/ml Brefeldin A (Sigma) was added to the cells and left for an additional 2 h at 37°C in a 5% CO₂ atmosphere. Surface or intracellular staining was performed using anti-mouse CD3-PerCP, CD4-Alexa Fluor 700, CD8-v450, NK1.1-PE (all BD Biosciences), IL-17A-FITC (eBioscience, San Diego, CA) and Live/Dead Fixable Aqua Dead Cell Stain Kit (Life Technologies, Carlsbad, CA). Stained cells were acquired using LSR-II Flow Cytometer and FACSDiva software (BD Biosciences), and subsequent analyses were conducted using FlowJo software (FlowJo, Treestar, Ashland, OR).

PMA (Sigma) and ionomycin (Sigma) were used for T cell activation. Briefly, PMA and ionomycin were incubated with PBMC for 6 hours at 37°C, GolgiStop was added after 1 hours of incubation. Cells were fixed and permeabilised by the BD Cytofix/Cytoperm Fixation/Permeabilisation Kit according to the manufacturer’s instruction before staining with the phenotype marker together with IL-17A FITC (eBiosciences) and IFN-γ PE-Cy7 (BD Biosciences). Samples were analyzed on a FACS Canto II machine with FACS Diva software evaluation.

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.