Prostin e2 pge2

Monocytes were isolated from PBMCs using BD IMag Anti-Human CD14 Magnetic Particles (557769; BD Biosciences). 6.0 × 10⁶ monocytes were seeded in a six-well plate in RPMI supplemented with 1% human serum (Sigma-Aldrich). Monocyte-derived DCs were generated adding 800 IU/ml of recombinant GM-CSF and 250 IU/ml of recombinant IL-4 (both from CellGenix). For EV isolation (see below), imDC were washed and 24 h later, the supernatant was harvested (10 ml). To generate maDCs, imDC cultures were supplemented for 24 h with LPS (100 ng/ml) or a maturation cocktail (200 IU/ml IL-1β, 1,000 IU/ml IL-6 (both from CellGenix), 10 ng/ml TNF (beromun; Boehringer Ingelheim), and 1 μg/ml Prostin E2 (PGE2; Pfizer). Subsequently, the cells were washed and EV supernatants (10 ml) were collected 24 h later for EV isolation.

Monocyte-derived DCs were generated as described previously (Heilingloh et al., 2014 (link)). In brief, peripheral blood mononuclear cells (PBMCs) were isolated from different healthy donors using a Lymphoprep gradient (Nycomed Pharma AS, Oslo, Norway) and afterward monocytes were separated using plastic adherence. By addition of 800 U/ml granulocyte macrophage-colony stimulating factor (GM-CSF; CellGenix, Freiburg, Germany) and 250 U/ml IL-4 (Milteny, Bergisch-Gladbach, Germany) monocytes differentiated to immature DCs. Maturation was induced by adding 10 ng/ml TNF-α (Beromun, Boehringer Ingelheim, Germany), 1 mg/ml prostin E2 (PGE2; Pfizer, NY, USA); 200 U/ml IL-1β (CellGenix), 40 U/ml GM-CSF, 1000 U/ml IL-6 (CellGenix), and 250 U/ml IL-4 to the medium. The maturation status was controlled by flow cytometry, and mDCs were used for further experiments.