Foxp3 apc clone 3g3

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).

For Tregs and effector-like T cells analysis, stimulated PBMCs were stained with CD4-FITC (0.6 μg/ml, clone SK3, BD Biosciences) and CD25-APC-Cy7 (2.5 μg/ml, clone M-A251, BD Biosciences) for 10 min at 4 °C in the dark. After incubation, cells were fixed, permeabilized and stained with FoxP3-APC (clone 3G3, Miltenyi Biotec) and with either Tbet-PE (clone REA102, Miltenyi Biotec) or GATA-3-PE (clone REA174, Miltenyi Biotec) or RORγt-PE (clone REA278, Miltenyi Biotec) for 30 min at 4 °C in the dark. Appropriate isotype controls were included for each sample.