Recombinant gm csf

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.