Cd19 bv480

Inguinal lymph nodes were collected 7 d post-MOG immunization. For flow cytometry of DCs, lymph nodes were cut into small pieces and incubated for 30 min at 37 °C in digestion mix: RPMI containing 0.5 mg ml⁻¹ of DNAse I and 0.5 mg ml⁻¹ of collagenase D. Cell suspensions were then filtered using 40-μm cell strainers. An antibody panel can be found in Supplementary Table 1. For tetramer staining, lymph nodes were dissociated by forcing through a 40-μm cell strainer. Cells were incubated for 3 h at 37 °C in RPMI containing 10% FCS in the presence of phycoerythrin (PE)-conjugated MOG tetramer (I-A(b) GWYRSPFSRVVH, 2.7 mg ml⁻¹) or control tetramer (I-A(b) PVSKMRMATPLLMQA, 2.7 mg ml⁻¹) (both obtained from the National Institutes of Health (NIH) tetramer core facility). After washing, cells were stained with anti-CD8 BUV395 (BD Bioscience, clone H35-17.2), T cell receptor (TCR)-β BUV737 (BD Bioscience, clone H57-597), CD19 BV480 (BD Bioscience, clone 1D3), CD4 PerCPCy5.5 (BD Bioscience, clone RM4-5) and CD11b PeCy7 (BD Bioscience, clone M1/70).

Whole blood or PBMC were stained with antibody mix comprising anti-human CD3-Bv421, CD4-PE, CD8-APC, CD14-APCH7, CD19-Bv480, CD20-PECy7, and CD45-FITC (clones UCHT1, SK3, SK1, MphiP9, SJ25C1, L27, 2D1, respectively) and 7-AAD (all from BD Biosciences). For whole blood, BD Trucount™ tubes were used according to the manufacturer’s instructions (Figure S1). Briefly, antibody mix was added followed by 50 μl whole blood by reverse pipetting. Tubes were vortexed and incubated for 15 min at room temperature in the dark. Erythrocytes were lysed by incubating with 450 μl 1xPharm Lyse™ (BD Biosciences), for 15min before analysing samples on a BD FACS Verse.