Monocyte-Derived Dendritic Cell Differentiation

Per well 1 × 10⁶ isolated monocytes where plated in 12-well plates (TPP) in 1 ml IMDM with 10% human AB serum (Karolinska University Hospital). Differentiation of monocytes to immature DC (iDC) was done using a fast protocol, in which iDC formation was established by 48 h of culture in the presence of 100 ng/ml GM-CSF (Peprotech) and 20 ng/ml IL-4 (Peprotech). Mature DC (maDC) were created by incubating iDC for an additional 18 h with one of the three following cocktails. The first was the gold standard [26 (link)]: 20 ng/ml tumor necrosis factor-α (TNF-α; Peprotech), 10 ng/ml interleukin-1β (IL-1β; CellGenix), 1000 U/ml interleukin-6 (IL-6; CellGenix), and 10 ng/ml PGE2 (SIGMA). Second, the alpha-type 1 polarizing cocktail [27 (link)]: 50 ng/ml TNF-α, 25 ng/ml IL1b, 3000 U/ml IFN-α (R&D Systems), 100 U/ml IFN-γ (Imukin^®, Boehringer Ingelheim), and 250 ng/ml polyinosinic:polycytidylic acid (poly I:C, Sigma-Aldrich). Finally, the COMBIG CCK Cocktail [28 (link)] was used: 10 ng/ml LPS (Sigma-Aldrich), 20 μg/ml Hiltonol (OncoVir), 2.5 ug/ml R848 (VacciGrade™, InvivoGen), and 1000 U/ml IFN-γ. Readout was performed by flow cytometry.