Peripheral Blood Mononuclear Cells and CSF

Peripheral blood mononuclear cells (PBMCs) were separated by density-gradient centrifugation (Lymphoprep, Axis-Shield, Oslo) according to the manufacturer's recommendations, then permeabilized and fixed for 15 minutes at room temperature in permeabilization buffer with 0.1% bovine serum albumin (BSA), and 4% paraformaldehyde in at 5 million cells per milliliter. Fixed, permeabilized cells were then washed with cell staining buffer (PBS 0.1% BSA, 0.01% NaN3, 2 mM EDTA), frozen on dry ice and stored at ‒80°C until labeling. CSF samples were centrifuged at 400g for 10 minutes at room temperature within 1 hour of lumbar puncture. Supernatants were aspirated, and pellets were resuspended, fixed, and permeabilized exactly like PBMCs.