Il 17 pe clone tc11 18h10

Splenocytes were collected from male BALB/c mouse (6–8 weeks old) spleens by standard techniques with PBS flushing. Splenocytes were gently scraped with a 1-mL syringe and passed through a 40-mm nylon cell strainer (BD Falcon, San Jose, CA, USA). Erythrocytes were removed using red blood cell lysis buffer (Biyuntian Biotech, Beijing, China). Splenic lymphocytes (2 × 10⁶ cells) were cultured in 6-well plates precoated with anti-mouse CD3 (10 μg/mL, clone 145–211) and anti-mouse CD28 (2 μg/mL, clone T2.5) antibodies (both from BioLegend, San Diego, CA, USA), and cocultured with or without bifidobacterial strains (1 × 10⁷ cfu) at a ratio of 5:1 splenocyte in RPMI-1640 medium (Gibco) supplemented with 10% FCS and 100 U/mL penicillin–streptomycin. Three days (72 h) later, cells were collected and stained with monoclonal antibodies CD4-PerCP-cy5.5 (clone GK1.5), CD4-FITC (clone GK1.5), Foxp3-PE (clone MF-14), CD25-APC (clone PC61.5) and IL-17-PE (clone TC11-18H10.1) (all from BioLegend). Fluorescent signals were acquired using a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed with FlowJo (Tree Star, Ashland, OR, USA) software.

To assess cytokine production on T cells infiltrating the CNS, immunized mice were sacrificed at peak disease and mononuclear cells from brain tissues were isolated as previously described (Garcia et al., 2013 (link)). T cells were stimulated for 4 h at 37°C and 5% CO₂ under the following conditions: cRMPI [RPMI 1640, Gibco; 10% Fetal bovine serum, Atlanta Biologicals; 1% Pen-Strep (Gibco), anti-CD3e (BioLegend), anti-CD28 (BD Pharmingen) and GolgiStop (BD Biosciences)]. For intracellular cytokine staining, the following reagents were used: Mouse Fc block (clone 2.4G2, BD Pharmingen); fixation/permeabilization buffer (Invitrogen); perm/wash buffer (BD Biosciences); cell staining buffer (BioLegend) and the following antibody cocktail: TCR-b V450 (clone H57-597, BD Horizon), CD4-APC-Cy7 (clone RM4-5, BioLegend), CD8a-PerCP/Cy5.5 (clone 53-6.7, BD Pharmingen), IL-17-PE (clone TC11-18H10.1, BioLegend) and IFN-γ-APC (clone XMG1.2, BioLegend) Samples were acquired using an ImageStreamX-Imaging Flow Cytometer-ISX-MKII (EMD Millipore) at the Cell Analysis Core, UTSA. Data analysis was performed using IDEAS software version 6.2.

The myelin oligodendrocyte glycoprotein peptide 35–55aa (MOG_35–55, MEVGWYRSPFSRVVHLYRN GK) was synthesized by SBS Genetech (San Francisco, CA). The purity of these peptides was in the range of 95.18%. This is MHC-II presented peptide interacting with CD4+ T cells. Complete Freund's adjuvant (CFA) and pertussis toxin (PTX) were from Sigma-Aldrich. Reagents purchased from BD Bioscience (San Jose, CA) were Mycobacterium tuberculosis H37Ra, fixation/permeabilization kit, and leukocyte-activation cocktail (LAC). Sulfo-SMCC and Keyhole Limpet Hemocyanin (KLH) were from Thermo Scientific (Waltham, MA). The following fluorescent antibodies were used: CD4-PerCP (clone RM4-5, BD); IL-17-PE (clone TC11-18H10.1, Biolegend (San Diego, CA)); Foxp3-APC (clone 3G3, Miltenyi Biotec (San Diego, CA)). Foxp3/transcription factor staining buffer set used for Foxp3 intracellular staining was from eBioscience (San Diego, CA). Mouse CD4+ T cell enrichment kits (EasySep) were purchased from Stem Cell Biotech (Vancouver, Canada). Carboxyfluorescein Succinimidyl Ester (CFSE) used for cell tracking and T cell proliferation assay was from Life Technology (Grand Island, NY).