, PE-Cy7 conjugated), anti-CD45RC (OX22, FITC conjugated), anti-CD28 (JJ319, biotin labeled), anti-CD71 (OX26, biotin labeled), antimouse V?11

, anti-CD25 (OX39, biotin labeled), and anti-MHC-II (OX6, biotin labeled)

, RT1-A a , ?2m, and Du51 peptides were refolded in 0.4 M L-arginine, 0.1 M Tris (pH 8), 2 mM EDTA, 5 mM reduced glutathione, and 0.5 mM oxidated glutathione for 5 days at 4°C. The solution was then concentrated, and the buffer was changed on a 10-kDa amicon membrane (Millipore). Folded MHC-peptide complexes were biotinylated with the BirA enzyme (Avidity) for 5 hours at 30°C and desalted on a Hiprep 26/10 desalting column (GE Healthcare). MHC-peptide complexes were then purified by anion exchange Q-sepharose chromatography. Biotinylation was tested by tetramerization with streptavidin (Sigma-Aldrich) at a molar ratio of 4:1. Tetramerization and staining. Tetramerization of RT1.A a /Du51 was performed at room temperature by the addition of streptavidin-PE (Jackson ImmunoResearch) or streptavidin-APC (BD Biosciences) at a 4:1 molar ratio, in 4 equal aliquots added at 15-minute intervals. Likewise, the control tetramer RT1.A a /MTF-E (ILFPSSERLISNR) was conjugated with streptavidin-BV421 (Biolegend) and represented 1.6 ± 0.7% of nonspecific staining among Du51-specific cells, For intracellular staining, cells were then biotin labeled for Foxp3 (eBioscience) using a BD Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer's instructions. All biotinylated mAbs were visualized using PerCP.Cy5.5 Streptavidin (BD Biosciences). A FACSCanto II cytofluorimeter (BD Biosciences) was used to measure fluorescence, and data were analyzed using FlowJo software, version 7.6.5 (Tree Star Inc.)

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