Fixable near ir dead cell stain

PBMCs were stained for surface markers and intranuclear IRF5. The cells were washed and stained with LIVE/DEAD fixable Near-IR dead cell stain (Invitrogen) and fluorochrome-conjugated surface antibodies (see Supplementary Table S2). Cells were fixed using 1% paraformaldehyde and subsequently permeabilized with 0.1% Triton X-100. Fixed cells were first incubated with an anti-IRF5 antibody (E7F9W, Cell Signaling Technology) for 20 min and then a secondary anti-rabbit-IgG AlexaFluor488 antibody (Cell Signaling Technology) for 15 min. Stimulated samples and unstimulated controls were stained for intracellular cytokines with fluorochrome-labeled antibodies. Samples were acquired within 12 h on a BD LSRFortessa II (BD Biosciences). Cells were analyzed using FlowJo 10.7 software (BD Biosciences) with the exclusion of doublet cells. mDCs were identified as LD−, CD3−, CD14−, CD19−, CD20−, HLA-DR+, CD11c+, and CD123− cells. pDCs were identified as LD−, CD3−, CD14−, CD19−, CD20−, HLA-DR+, CD11c−, and CD123+ cells. Background staining was assessed for every sample by staining without the primary anti-IRF5 antibody (isotype control).