Fc block

Tetramer enrichment was performed as previously described (32 (link)). In brief, spleen and peripheral lymph nodes (inguinal, lumbar, mesenteric, cervical, axillary and brachial) were harvested and processed into a single cell suspension by passing through a 100μm strainer. Cells were washed in cold 1X HBSS (Cellgro) and resuspended with 4μg/mL of tetramer PE conjugated NFM:I-A^b and/or APC conjugated MOG:I-A^b (NIH tetramer core (33 (link), 34 (link))) in Fc block (heat killed mouse and rat serum, Sigma-Aldrich) at a volume 2× the pellet volume. After 1hr at room temperature, cells were wash in cold FACS wash (0.1% BSA, 0.05% NaN3, 1x PBS) and resuspend in 200μL FACS wash plus 50μL of anti-PE and/or anti-APC beads (Miltenyi Biotec) and incubated for 30 min. on ice. Cells were then washed and enriched on a LS column (Miltenyi Biotec). Unbound and column bound cell numbers were determined with AccuCheck microbeads (Invitrogen) alongside with cell surface marker characterization performed with flow cytometry (LSR II, BD) and FlowJo software (Treestar). Antibodies used included; CD3ε-FITC (145-2C11, BD Pharmingen), CD11b- PerCP -Cy5.5 (M1/70, BD Pharmingen), CD11c-PerCP-Cy5.5 (HL3, BD Pharmingen), CD19- PerCP -Cy5.5 (1D3, Tonbo Biosciences), CD4-Brilliant Violet 510 (RM4-5, BioLegend), CD8-Brilliant Violet 785 (53-6.7, BioLegend), CD44-Alexa Fluor 700 (IM7, eBioscience).