Donkey anti rabbit alexafluor 647

Staining was performed on cells isolated from EAE mice or following in vitro treatments. Cells were stained with a viability dye (Biolegend, SanDiego, CA) in 1X PBS for 20 min and then washed in PBS. They were then incubated with Fc block for 15 min (and cells for intracellular IgG staining were also incubated with anti-IgG to block extracellular IgG), followed by fluorescentlyconjugated antibody staining for 30 min. Extracellular antibodies were all obtained from Biolegend: CD19 (PE/Cy7), FasL (PE), PD-L1 (APC), IgM (FITC) and IgD (PacBlue). The cells were washed in flow cytometry buffer (FCM; 0.1% bovine serum albumin in Hank’s buffered saline solution) and then fixed with Cytofix (BD Biosciences, San Jose, CA) for 15 min. For intracellular IgG or Cyp1a1, the cells were permeabilized and incubated with IgG (APC) or Cyp1a1 (purified; Abcam, Cambridge, MA) for 1 hr. Detection of Cyp1a1 also required incubation with a conjugated secondary prior to detection (donkey anti-rabbit AlexaFluor 647; Biolegend) for 30 min. After staining, the cells were washed and resuspended in FCM and analyzed on an ACEA Novocyte (San Diego, CA). Fluorescence minus one (FMO) controls provided guidance on gate setting. Data analysis was done with NovoExpress software.