For surface staining of BDCA-4 and CD123, pDCs were washed with PBS and incubated for 30 min at 4°C with a viability stain (Zombie Aqua Fixable Viability Kit, BioLegend). Cells were washed with magnetic-activated cell sorting (MACS) buffer (PBS, 2% fetal bovine serum, and 2 mM EDTA) and incubated for 30 min at 4°C with the appropriate antibodies or with the corresponding isotype control antibodies (5 mg/ml each) in MACS buffer containing Fc receptor blockers (BD Biosciences, San Jose, CA).

For intracellular IFN staining, cells were treated with brefeldin A (BFA) at 1 μg/ml for the last 12 hours. Cells were fixed using 2% paraformaldehyde for 10 min at room temperature. After washing the cells with MACS buffer containing 0.1% saponin (Sigma-Aldrich), cells were stained for 1 hour at 4°C in MACS buffer containing 0.1% saponin.

For intranuclear pIRF7 staining, cells were fixed with Fix Buffer I and permeabilized with Perm Buffer III (BD Biosciences), following the manufacturer’s instructions. After washing the cells with MACS buffer, cells were stained for 1 hour at 4°C.

Flow cytometry analyses were performed on a BD FACSCanto II flow cytometer using flow cytometry (Diva software, BD Biosciences, San Jose, CA). FlowJo software (Tree Star, Ashland, OR) was used to analyze data.

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