Isolation and Analysis of PBMCs

Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA anticoagulated blood. Blood and phosphate-buffered saline supplemented with 0.5% bovine serum albumin (PBS/BSA) were mixed at a 1:1 ratio and 35 ml were layered onto 15 ml Ficoll-Paque PLUS gradient (GE Healthcare). PBMCs were isolated according to the manufacturer’s protocol and stored in liquid nitrogen until the analysis.
For analysis batches of 20–35 samples were thawed and washed in RPMI 1640 cell medium (Gibco). In each batch, a mix of samples from healthy controls and MS/NMOSD patients were included. Cells were stained with antibodies against CD14 (DRFZ, clone TM1), CD169 (SIGLEC1, clone 7–239, BioLegend Cat# 346004, RRID:AB_2189029) and a dead cell stain (eBioscience Fixable Viability Dye eFluor 780). For each batch, a fluorescence minus one (FMO) control for SIGLEC1 was measured and FMO measurements remained stable over multiple batches. The samples were acquired on a FACSCanto cytometer (BD Biosciences) and all cytometry experiments were performed according to published standards^{26 (link)}. Using the FlowJo software (Version 10.4.1 for Mac, FlowJo LLC), we identified monocytes and excluded doublets based on scatter parameters. We then gated on CD14^high, living cells and analysed the median fluorescence intensity (MFI) for SIGLEC1 (Fig. 1a).