Generation of Monocyte-Derived Dendritic Cells

Human monocyte-derived DCs were generated as previously described^{31 (link)}. In brief, human peripheral blood mononuclear cells (PBMCs) were harvested by density gradient centrifugation using Biocoll (Biochrom AG). CD14⁺ primary human monocytes were further purified from PBMCs by CD14⁺ microbeads (Miltenyi Biotech) using a magnetic cell sorting system. Primary monocytes were differentiated into monocyte-derived dendritic cells (moDCs) by treatment with 800 U/ml human recombinant GM-CSF and 1,000 U/ml human recombinant IL-4 (Miltenyi Biotech). At day six cell surface marker expression (Fig. S1A) and phenotype of immature moDCs was monitored prior to performing conidia interaction studies.