Recombinant human granulocyte macrophage colony stimulating factor gm csf

Human peripheral blood mononucleated cells (PBMCs) of healthy donors were isolated from leucocyte reduction system chambers (LRSCs) using density centrifugation as described previously [30 (link)]. For the generation of monocytes, cells were seeded in DC medium consisting of RPMI1640 + 1% penicillin/streptomycin/glutamate + 1% heat-inactivated AB serum + 1% HEPES after removing the non-adherent fraction (NAF). NAF was cryopreserved and stored at −80 °C for isolation of T cells for allogeneic MLR or for antigen-specific T-cell priming.
For the generation of DCs from PBMCs, the adherent cell fraction was cultured for 4 days in DC medium supplemented with 800 IU/mL (day 0) or 400 IU/mL (day 3) recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) and 250 IU/mL (day 0 and 3) recombinant IL-4 (both Miltenyi Biotec, Bergisch Gladbach, Germany). On day 4, maturation of DCs was induced when indicated by adding a maturation cocktail consisting of 1000 IU/mL IL-6 (Miltenyi Biotec), 200 IU/mL IL-1β (Cell Genix, Freiburg, Germany), 10 ng/mL tumor necrosis factor α (TNF-α; Peptrotech, Hamburg, Germany), and 1 µg/mL prostaglandin E2 (PGE2; Santa Cruz, Dallas, TX, USA) for 24 h before cells were transduced with adenovirus.

Endotoxin-free reagents and plastic materials were used in all experiments. RPMI-1640, phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco (Invitrogen, Argentina). Twenty-four-well flat bottom polystyrene plates were purchased from Jet-biofil (AP Biotech, Buenos Aires, Argentina) while 96-well U-bottom plates and half-area 96-well ELISA were obtained from Greiner Bio One (GBO, Buenos Aires, Argentina). Ficoll-Paque PLUS and Percoll were obtained from GE Healthcare Life Sciences (Embiotec, Buenos Aires, Argentina). Recombinant human IL-4 and recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) were obtained from Miltenyi Biotec (Lab Systems, Buenos Aires, Argentina). Lipopolysaccharide (LPS) from Escherichia coli was purchased from Sigma-Aldrich (Merck, Argentina).

As previously reported, PBMCs and human-derived macrophages (MDMs) were isolated or differentiated (37 (link), 38 (link)). Briefly, PBMCs were enriched from whole blood by density gradient using Histopaque-1077 (Sigma-Aldrich, UK). Cells were washed twice in PBS and resuspended in Complete Roswell Park Memorial Institute Medium (cRPMI-1640) culture medium (RPMI 1640 with 2 mM glutamine; Gibco, Thermo Fisher Scientific, USA), 10% human serum (Sigma-Aldrich, USA), 10 U/mL penicillin/streptomycin, and 10 mM HEPES (Thermo Fisher Scientific, the Netherlands). Monocytes (CD14⁺) were isolated from PBMCs by positive selection using magnetically labeled CD14 Microbeads (Miltenyi Biotec). The isolated monocytes were then resuspended in cRPMI medium with 20 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF, Miltenyi Biotec), and cells were seeded at a concentration of 1 × 10⁶ cells/mL in 24-well and 48-well plates (Corning Inc., South Korea) for 7 days. The culture medium was refreshed on the fourth day of incubation. Before infection, the acquisition of macrophage morphology was confirmed by visualization in a BX61 microscope (Olympus).