Cytoflex s ow cytometer

To investigate changes in neutrophil subsets, 5*10 5 immediately xed cells were stained with anti-CD66b (1:5), anti-CD62L (AF647, 1:5; BioLegend), anti-CD16 (PE-Cy7, 1:5; BioLegend) and propidium iodide (PI, 1:5; BD Bioscience) for 15 minutes (min), RT, in the dark. Phosphate buffer saline (PBS, Gibco) was added to each sample to stop the staining procedure. All samples were analysed using a CytoFLEX S ow cytometer (Beckman Coulter). Single, live CD66+ cells were analysed according to their expression level of CD62L and CD16. Gates were de ned in the following manner: Neutrophils with high CD62L and high CD16 expression were de ned to be mature, low CD16 and high CD62L expression as newly released and cells low in CD62L, regardless of the CD16 expression levels, were de ned as activated. Raw data was analysed using FlowJo V10 (FlowJo, LLC).

Stimulation of 10 6 isolated cells in 90 µL complete RPMI was performed using IO (Thermo Fisher) and PMA (Sigma Aldrich), in the same concentrations as for neutrophil activation. After incubation for 3h (and mixing every 30 min) at 37°C, samples were xed with PFA ( nal concentration: 1%; Thermo Fisher). Samples were blocked with PBS (Gibco) containing 2% bovine serum albumin (Sigma Aldrich; blocking buffer) rst and then stained with a primary anti-H3Cit antibody (1:180) for 20 minutes at RT in the dark. After incubation, blocking buffer was added and samples were then centrifuged at 3200 x g for 10 min at 4°C. Supernatant was discarded and secondary antibody (Alexa Fluor 647 goat anti-rabbit IgG, 1:10000; Invitrogen) in combination with anti-CD66b antibody (1:40) was added for 20 min in the dark at RT. After a further washing step using blocking buffer, PI (1:50; BD Bioscience) was added to all stained samples. One sample per cell fraction was left unstained; another was stained with only the secondary antibody to eliminated false positive signals due to unspeci c antibody binding. As a gating strategy, dead CD66b+ singlets were analysed for their H3Cit expression. The gates for H3Cit+ cells were de ned via the secondary only samples from the respective group. Samples were analysed using the CytoFLEX S ow cytometer (Beckman Coulter®) and FlowJo V10 (FlowJo, LLC).

Cells were resuspended in 100 ml FCB, blocked with 0.5 ml TruStainFcX (anti-mouse CD16/32 FcR block, Biolegend) for 5 min at room temperature, and stained for 30 min at 4°C with: FITC-anti-CD45 (1:1000, Biolegend), PE-Cy7-anti-CD4 (1:200, eBioscience), PerCP-Cy5.5-anti-CD8 (1:200, Biolegend), PE-anti-B220 (1:200, Biolegend), APC-anti-MHCII (1:200, eBioscience), and APC-eFluor780-anti-CD11b (1:200, eBioscience) (Supplemental Table 1). Cells were then xed in 1% PFA for 1 h. Finally, cells were resuspended in 500 ml FCB, labelled with DAPI to exclude cell debris and analyzed with a CytoFLEX S ow cytometer (Beckman-Coulter) equipped with CytExpert software (Beckman-Coulter).
Quanti cation of IC100 in Tissues. IC100 was quanti ed in brain, spinal cord, liver and spleen at 35 days post-induction (dpi) of EAE using a proprietary assay developed by In amaCORE, LLC using Meso Scale Technology. Protein lysates were obtained as described in [29] . The assay was read using the QuickPlex SQ 120 instrument (Meso Scale Diagnostics, Maryland).