αcd16 32 93

Ex vivo-obtained splenic cells, CNS-infiltrating cells, and in vitro-differentiated T cells were analyzed by staining for extra- and intracellular markers. Dead cells were excluded by a fixable viability dye eFluor®780 (0.2 μl/test, eBioscience). Nonspecific Fc-mediated interactions were blocked by the addition of 0.5 μl αCD16/32 (93, eBioscience). For surface staining, the cells were treated with the respective fluorochrome-conjugated antibodies: αCD4-FITC (RM4-5, eBioscience) and αCD25-APC (PC61.5, eBioscience). For intracellular cytokine staining, cells were stimulated for 4 h with ionomycin (1 μM, Sigma-Aldrich, St. Louis, MO) and PMA (50 ng/ml, Sigma-Aldrich) in the presence of monensin (2 μM, eBioscience), fixed with 1% paraformaldehyde and made permeable by saponin or Fix/Perm buffer (eBioscience) treatment. Intracellular cytokines were stained with the respective fluorochrome-conjugated antibodies: αFoxP3-PE (FJK-16s, eBioscience), αIFNγ-APC (XMG1.2, eBioscience), αIL-17A-PE (eBio17B7, eBioscience), IL-4-PE (11B11, BioLegend), and GATA3-PerCP/eFluor710 (TWAJ, eBioscience). Probes were measured with a flow cytometer (FACSCantoII, BD Biosiences).

As previously described (40 (link)), dead cells were excluded by the fixable viability dye eFluor^®780 (0.2 μl/test, eBioscience). Nonspecific Fc-mediated interactions were blocked by addition of 0.5 µl αCD16/32 (93, eBioscience). For surface staining, cells were treated with the respective fluorochrome-conjugated antibodies: αCD4-BV510 (RM4-5, Biolegend), αCD11c-FITC (HL3, BD Biosciences), αCD25-APC (PC61.5, eBioscience), αCD80-APC (16-10A1, Biolegend), αCD86-PerCP/Cy5.5 (GL-1, Biolegend), and αMHCII-BV510 (M5/114.15.2, Biole-gend).
For intracellular cytokine staining, cells were stimulated for 4 h with ionomycin (1 µM, Sigma-Aldrich) and PMA (50 ng/ml, Sigma-Aldrich) in the presence of monensin (2 µM, eBioscience), fixed with 1% paraformaldehyde and made permeable by saponin buffer treatment. Intracellular cytokines were stained with the respective fluorochrome-conjugated antibodies: αFoxp3-PE (FJK-16s, eBioscience), αIFNγ-APC (XMG1.2, eBioscience), and αIL-17A-PE (eBio17B7, eBioscience). Samples were measured with a flow cytometer (FACSCantoII, BD Biosciences).