Monocyte, dendritic cell, and macrophage isolation

PBMCs were obtained by density gradient centrifugation and monocytes were subsequently purified by immunomagnetic separation using anti-CD14–conjugated magnetic microbeads (Miltenyi Biotec) according to the manufacturer’s protocol and as previously published (23 (link)). Briefly, monocytes were cultured for 6 days in tissue culture plates in complete medium (RPMI 1640 supplemented with 10% heat-inactivated, endotoxin-free FBS, 2 mM L-glutamine, penicillin, and streptomycin; all from Gibco, Thermo Fisher Scientific) in the presence of 50 ng/mL GM-CSF and 20 ng/mL IL-4 (Miltenyi Biotec). Untouched peripheral blood cDC1 and cDC2 (cDCs) and pDCs were obtained from PBMCs after negative immunomagnetic separation with the Myeloid Dendritic Cell Isolation kit (Miltenyi Biotec) and the Plasmacytoid Dendritic Cell Isolation kit II (Miltenyi Biotec), respectively. pDCs were cultured in completed RPMI medium with 20 ng/mL IL-3 (Miltenyi Biotec). RAW264.7 cells were purchased from American Type Culture Collection (ATCC) and cultured in DMEM complemented with 10% FBS.