Siglech ebio440c

For FACS analysis as well as FACS sorting of single-cell suspensions, cells were first incubated with CD16/CD32-specific antibody (2.4G2; BD) for 10 min to block non-specific Fc-receptor interactions. A live death staining was performed using zombie aqua (eBioscience) according to the manufacturer’s instructions. To differentiate between DC subsets, cells were stained for 15 min at 4°C with combinations of antibodies specifically binding to CD11b (M1/70.15; Invitrogen), CD11c (HL3; BD), Siglec-H (eBio440c; eBioscience), CD69 (H1.2F3; BD), Clec9a (42D2; eBioscience), CD172 (SIRPα) (P84; Biolegend) and CD24 (M1/69; Biolegend). The cells were subsequently washed with 1 mL FACS buffer (PBS, 1% FCS) and then re-suspended in FACS buffer supplemented with 3% paraformaldehyde (PFA). Samples were measured using a FACS LSR II, and data were analyzed with FlowJo 7.6.5 software (TreeStar). Dead cells as well as cell duplets were excluded prior to subsequent analysis. DC subsets were sorted using the FACS Aria or FACS XDP and sorting efficiency ranged between 71.9–86.2% for the pDC, 81.4–94.4% for the CD11b-like DC and 44.54–83.2% for the CD8α-like DC.

BMDCs were obtained by flushing bone marrow of femurs and tibias of CF-1 mice as previously described with minor modifications^{74 (link)}. Cells were plated at 1 × 10⁶/ml with RPMI 1640 supplemented with 5% heat-inactivated fetal bovine serum, 100 U/ml penicillin/streptomycin, 10 μg/ml gentamicin and 2 mM l-glutamine, (all from Thermo Fisher) and in presence of 100 ng/ml Flt3L (R&DSystems) at 37 °C in 5% CO₂ for 6 days. Finally, DC-population was characterized by flow cytometry using fluorescence-conjugated monoclonal antibodies (mAb) directed against CD11c (HL3), Flt3 (A2F10), Clec9a (42D2), CD172a (P84), CD11b (M1/70), CD3 (145-2C11), CD45R/B220 (RA3-6B2), SiglecH (eBio440c), and CD24 (M1/69) (eBiosciences). Approximately 70–80% of the cells were CD11c⁺.