Phenotype of BMDC and splenic DC subsets
The phenotype of BMDC was determined before and after 24 hour culture in the presence of 100 ng/mL LPS. The cells were incubated in PBS with 0.1% NaN3, 1% BSA and 10% normal rabbit serum for 20 minutes on ice, washed and incubated for 30 minutes on ice with Alex Fluor-labeled anti-CD11c (MCD11C20, CALTAG) in combination with PE- anti-CD40 (3/23, BD Biosciences), PE-anti-CD80 (16-10A1, eBioscience), PE-anti-CD86 (P03.1, eBioscience), PE-anti-CD14 (Sa2.8, eBioscience) and biotinylated anti-TLR4 (BioLegend). The cells incubated with biotinylated anti-TLR4/MD-2 were washed twice and incubated for 30 minutes on ice with avidin-PE. To isolate splenic DC, spleens were collagenase digested and subject to Percoll gradient centrifugation. The bands at the 35–55% interface were collected to stain with PE-anti-CD11c (HL3, BioLegend), APC-anti-PDCA-1 (927, BioLegend) and FITC-anti-CD8α (53–6.7, BD BioScience). The cells were washed twice, fixed in 2% paraformaldehyde, and stored at 4°C until analysis. Flow cytometry was performed in an Excel (Coulter) instrument. Dead cells were omitted from the analysis by gating on forward and 90° light scatter, and 10,000 cells were analyzed by FlowJo software.