Cd4 percp clone rm4 5

Mice were sacrificed and subsequently, blood and spleen were harvested of six mice per group. White blood cells were obtained as described above. 300,000 cells per sample were stained with the appropriate FACS antibodies. The following antibodies were used: CD11b-FITC (clone M1/70), CD11c-PE (clone N418), CD30L-PE (clone RM153), CD4-PerCP (clone RM4–5; BD Biosciences), CD40-FITC (clone HM40-3), CD8-PerCP (clone 53-6.7; BD Biosciences), CD80-PE (clone 16-10A1), CD86-APC (clone GL1), FoxP3-APC (clone FJK-16s), Gata-3-PE (clone TWAJ), Ki-67-FITC (clone SolA15), Ly-6G-APC (clone 1A8; BD Biosciences), MHCII-FITC (clone AF6-120.1), OX40L-PE (clone RM124L), PD-L2-APC (clone MIH18), RORγt-PE (clone AFKJS-9), and T-bet-Alexa Fluor 647 (clone eBio4B10). All antibodies were purchased from eBioscience, unless stated otherwise. For intracellular staining, cells were fixed and permeabilized according to the manufacturer’s protocol (eBioscience). FACS analysis was performed on the FACS Canto II and data were analyzed using FACS Diva software (BD Biosciences).

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).