Conventional Bone Marrow-Derived Dendritic Cells Contribute to Toll-Like Receptor-Independent Production of Alpha/Beta Interferon in Response to Inactivated Parapoxvirus Ovis

Mice：C57BL/6J  wild-type  mice， Bone  marrow  from mice 8 to 16 weeks old was used for all experiments.

Cell culture： Murine  bone  marrow  cells  from  C57BL/6J  wild-type, cultivated for 7 days at 37°C in a humidified atmosphere containing 5% CO₂  in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 U of penicillin/ml, 100 μg of streptomycin/ml, 50 μM β-mercaptoethanol (Sigma, Taufkirchen, Germany), 0.2% vitamins, essential and nonessential amino acids, and 1% murine Flt3L-containing supernatant generated in our laboratory.

Cell purification： Where indicated, cells were purified prior to stimulation by high-gradient cell separation (MACS; Miltenyi Biotec, Bergisch-Gladbach, Germany). Either CD11b⁺ BM-cDC or B220⁺ BM-pDC were removed with fluorescein  isothiocyanate  (FITC)/phycoerythrin  (PE)-labeled  anti-mouse-CD11b (clone  M1/70.15;  Caltag  Laboratories,  Burlingame,  CA)  or  anti-mouse-B220
(clone RA3-6B2; Caltag Laboratories), respectively, followed by incubation with an anti-FITC/PE-antibody coupled to magnetic beads (Miltenyi Biotec) according to the manufacturer’s instructions using LD MACS columns and the Vario-MACS  system  (Miltenyi  Biotec).  The  success  of  depletion  was  confirmed  by fluorescence-activated cell sorting (FACS) analysis prior to stimulation. Purified
BM-pDC contained at most less than 4% BM-cDC.