Dendritic cells (DCs) were isolated from the spleens or inguinal lymph nodes of HHD-I mice by positive selection using CD11c MicroBeads (Miltenyi Biotech GmbH) and cultured in mouse DC growth medium (RPMI 1640 with 10% FCS, penicillin, streptomycin, non-essential amino acids, sodium pyruvate, L-glutamine, and β-mercaptoethanol) as described earlier.59 (link),60 (link) For subset analysis, purified DCs were stained at 4 °C for 30mins with different staining panels, including CD11c (AF700) (eBioscience), CD8 (PerCP) (BD Biosciences, New Jersey, MD), B220 (PE) (BD Biosciences), CD103 (FITC) (eBioscience), CD326 (PE-Cy7) (eBioscience), and DEC205 (APC) (eBioscience). Cells were washed and acquired on FACS Canto II to determine exact cell numbers.
To determine the inflammatory cytokine response, CD11c+DCs were incubated with MPL and/or CpG for 20 h. Supernatants were the collected and levels of IL-6, IL-12p70, TNF, and IFNα were assessed using an ELISA assay (eBioscience). Expression of IL-12p70 in CD11c+ DCs was also analyzed by intracellular staining. Briefly, these cells were incubated with AF700-conjugated anti-CD11c and FITC-conjugated anti-PDCA-1 monoclonal antibodies (eBioscience), then fixed and permeabilised, and stained with PE-conjugated anti-IL-12p70 (BD Biosciences). Cells were acquired on a FACSCanto II and analyzed using FlowJo software.
To determine the inflammatory cytokine response, CD11c+DCs were incubated with MPL and/or CpG for 20 h. Supernatants were the collected and levels of IL-6, IL-12p70, TNF, and IFNα were assessed using an ELISA assay (eBioscience). Expression of IL-12p70 in CD11c+ DCs was also analyzed by intracellular staining. Briefly, these cells were incubated with AF700-conjugated anti-CD11c and FITC-conjugated anti-PDCA-1 monoclonal antibodies (eBioscience), then fixed and permeabilised, and stained with PE-conjugated anti-IL-12p70 (BD Biosciences). Cells were acquired on a FACSCanto II and analyzed using FlowJo software.