Lsrfortessa cytometer

Plasma samples were screened for autoantibodies against IFN-α, IFN-β, IFN-ω and IFN-γ in a multiplex particle-based assay⁸³ , in which differentially fluorescent magnetic beads were covalently coupled to recombinant human proteins (2.5 μg/reaction). Beads were combined and incubated for 30 minutes with diluted plasma samples (1 to 100 dilution). Beads were then washed and incubated with PE-labeled goat anti-human IgG (1 ug/mL) for an additional 30 minutes. Beads were washed again, resuspended in assay buffer, and analyzed on a BioPlex X200 instrument. Plasma samples with a fluorescence intensity > 1,500 were tested for blocking activity. The blocking activity of autoantibodies was determined by assessing STAT1 phosphorylation in healthy control cells following stimulation with the appropriate cytokines in the presence of 10% healthy control or patient plasma. Surface-stained healthy control PBMCs were cultured in serum-free RPMI medium with 10% healthy control or patient plasma and were either left unstimulated or stimulated with 10 ng/mL of IFN-α, IFN-β, or IFN-ω or 400 units/mL of IFN-γ for 15 minutes at 37°C. Cells were fixed, permeabilized, and stained for intranuclear pSTAT1 (Y701). Cells were acquired on a BD LSRFortessa cytometer, gated on CD14+ monocytes, and analyzed with FlowJo software.