Blood dendritic cell isolation kit 2

Circulating dendritic cells (DCs) were isolated from the blood of healthy donors using the Blood Dendritic Cell Isolation Kit II (Miltenyi Biotec). Purity was assessed as more than 80% by flow cytometry using exclusion staining based on CD3, CD14 and CD20.

Monocyte-derived dentritic cells (Mo-DC) were generated as described previously.^{13 (link)} Blood DC from healthy donors and patients were isolated using Blood Dendritic Cell Isolation Kit II (Miltenyi Biotech). Where indicated, HLA-DR⁺ blood DC were further sorted either as CD1c⁺ conventional DC (cDC) or CD303⁺ plasmacytoid DC (pDC).

Normal human skin samples were obtained as the discarded products of dermatologic surgery. Subcutaneous fat was removed, and remaining tissue was washed with PBS. The dermal layer was scratched as described above and incubated with Dispase II (1 mg/ml; Gibco) overnight at 37°C. Epidermis and dermis were separated with forceps. Epidermal sheets were chopped with scissors and incubated with Liberase (0.3 mg/ml; Roche) at 37°C for 1.5 h LCs were isolated from epidermal cell suspension by using a CD1a isolation kit (Miltenyi Biotec). Blood DCs were isolated by using Blood Dendritic Cell Isolation kit II (Miltenyi Biotec). DCs were primed with recombinant IL-23 (100 ng/ml; R&D Systems) for 24 h. After extensive washing, DCs were co-cultured with allogeneic naive CD4 T cells (Miltenyi Biotec) in the presence of anti-CD3/CD28 beads (Miltenyi Biotec) for 5 d. Cell-free supernatants were used to detect IL-22, IL-17A, IL-13, and IFN-γ by ELISA kit (eBioscience).