Isolation of Distinct Dendritic Cell Subsets

Isolation of slanDCs was performed as described previously [6 (link)]. PBMCs were incubated with M-DC8 hybridoma supernatant containing 10 μg/ml of antibody and additional rat anti-mouse IgM paramagnetic microbeads (Miltenyi Biotec). Cells were sorted on two columns via the autoMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany). CD1 + DC were sorted by depletion of CD19+ cells first, followed by positive selection of BDCA1+ using immunomagnetic separation according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). CD14+ monocytes were isolated by positive selection, and CD4+ T cells, CD8+ T cells, and naive CD45RA + CD4+ T cells were isolated by depletion using immunomagnetic separation (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the isolated cell populations was >95% as always assessed by flow cytometry afterwards.