Buffy coats from anonymized healthy donors were obtained from the “DRK‐Blutspendedienst—Ost” (Berlin, Germany). PBMC were isolated by density gradient centrifugation using Ficoll (Biochrom, Berlin, Germany; now Merck Millipore). pDC were prepared using the Human Diamond pDC Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. B cells were isolated from PBMC using the CD19 MicroBead Kit (Miltenyi Biotec).
Cells were cultured in complete medium (RPMI1640 [Lonza, Basel, Switzerland] with 2 mM UltraGlutamine [Lonza] supplemented with 10% (v/v) fetal calf serum [Linaris, Dossenheim, Germany], 100 U/mL Penicillin and 100 μg/mL Streptomycin) in flat‐bottom plates (six million PBMC/mL, two million B cells/mL) or 96‐well round bottom plates (0.25 million pDC/mL, in the presence of 10 ng/mL recombinant IL‐3 [PeproTech, Hamburg, Germany]). B18R (eBioscience, Frankfurt/M, Germany), a vaccinia virus‐encoded receptor with specificity to type I interferons, was used at a final concentration of 0.5 μg/mL.
Cells were cultured in complete medium (RPMI1640 [Lonza, Basel, Switzerland] with 2 mM UltraGlutamine [Lonza] supplemented with 10% (v/v) fetal calf serum [Linaris, Dossenheim, Germany], 100 U/mL Penicillin and 100 μg/mL Streptomycin) in flat‐bottom plates (six million PBMC/mL, two million B cells/mL) or 96‐well round bottom plates (0.25 million pDC/mL, in the presence of 10 ng/mL recombinant IL‐3 [PeproTech, Hamburg, Germany]). B18R (eBioscience, Frankfurt/M, Germany), a vaccinia virus‐encoded receptor with specificity to type I interferons, was used at a final concentration of 0.5 μg/mL.