IL-15 DCs were prepared as per our previously reported rapid DC culture protocol [11 (link), 12 , 14 (link)]. Briefly, monocytes were seeded in Roswell Park Memorial Institute medium (RPMI; Life Technologies) supplemented with 2.5% heat-inactivated human AB serum (hAB; Invitrogen, Merelbeke, Belgium) at a final concentration of 1.0–1.2 × 106 cells/mL. Differentiation was induced with 800 IU/mL granulocyte macrophage colony-stimulating factor and 200 ng/mL IL-15 (Immunotools, Friesoythe, Germany). A TLR-activating maturation cocktail, comprising R848 (3 μg/mL; Alexis Biochemicals, San Diego, USA), tumor necrosis factor-α (2.5 ng/mL), interferon-γ (250 ng/mL; Immunotools) and prostaglandin E2 (1 μg/mL; Pfizer, Puurs, Belgium), was added after 24–48 hours of differentiation for 18–20 hours. All components were bought from Invitrogen, unless stated otherwise. Control 7-day IL-4 DCs from the same blood donors were prepared as previously described in detail [11 (link)]. All subsequent experiments were performed using the obtained activated IL-15 DCs and IL-4 DCs. For the collection of x-hour wash-out supernatant, mature DCs were harvested, washed thoroughly and resuspended in fresh medium, RPMI + 2.5% hAB, at a concentration of 1 × 106 cells/mL. After x hours of culturing in low absorbing polypropylene tubes, cell-free supernatant was collected and frozen at −20°C until further use.
Free full text: Click here
