Isolation and Culture of Murine Plasmacytoid Dendritic Cells

For DC isolation, spleens or thymuses were cut and digested with collagenase III and Dnase I, followed by light-density separation and then immune-magnetic bead depletion using the procedure described elsewhere (23 (link)). For generation of Fms-like tyrosine kinase 3 (Flt3) ligand (FL)-cultured bone marrow-derived pDCs (24 (link), 25 (link)) in vitro, total bone marrow cells were cultured for 7–9 days in culture medium consisting of RPMI1640 medium supplemented with essential and nonessential amino acids, 5.5 × 10⁻⁵mol/L 2-mercaptoethanol, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% FBS (Gibco) with 100 ng/mL recombinant murine FL (Peprotech). Dead cells were excluded by 7-AAD viability staining (eBioscience). DCs were stained with combinations of mAbs to CD11c (N418), SiglecH (eBio440C), B220 (RA3-6B2), CD172a (P84), CD8a (53-6.7), and CD24 (M1/69). CD11c^intSiglecH⁺B220⁺ were used to gate pDCs population. The antibodies were purchased from eBioscience (San Diego, CA, USA) or Biolegend (San Diego, CA, USA). Cells were analyzed with a LSRII flow cytometer (BD) or sorted with a FACSAria III machine (BD). FACS data were analyzed and displayed by FlowJo software (Tree Star).