Recombinant human granulocyte macrophage colony stimulating factor gm csf

Buffy coats were bought from the Blood Transfusion Clinic at the Karolinska University Hospital, and monocytes were isolated from the buffy coats using commercial kits (RosetteSep Human Monocyte Enrichment Cocktail solution, Stemcell, Grenoble, France) and density gradient centrifugation on lymphoprep (Fresenius Kabi Norge AS, Oslo, Norway). To generate DC, the purified monocytes were cultured in RPMI 1640 medium containing GlutaMAX (Invitrogen Gibco, Paisly, UK) supplemented with 10% fetal bovine serum (HyClone,, Logan, UT, USA), 100 µg/mL streptomycin and 100 U/mL penicillin (Invitrogen Gibco), 250 ng/ml of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (R&D systems or PeproTech, London, UK) and 6.5 ng/ml of recombinant human IL-4 (R&D systems). RPMI 1640 is described above, but without GM-CSF and IL-4 is referred to as complete RPMI, whereas RPMI 1640 with no supplementary agents is referred to as incomplete RPMI. On day 3 of culture, half of the medium was replaced with fresh complete RPMI containing IL-4 and GM-CSF. On day 6 or 7 the cells were harvested and washed with incomplete RPMI medium. The cell population used for the experiments described below typically contained >70% CD14− CD1a+ live cells, assessed by flow cytometry (data not shown).

Freshly purified monocytes were cultured in complete RPMI 1640 media (1% L-glutamine (R&D Systems, Minneapolis, MN, USA), 10% heat-inactivated FBS, 1% penicillin-streptomycin (PS), 1% non-essential amino acids and 1 mM sodium pyruvate (all from Sigma-Aldrich, St. Louis, MO, USA)). For monocyte-derived cultures, cells were plated in 24 wells plate (1×10⁶ cells per well) in complete RPMI 1640 medium with 75 U/ml IL-4 and 1000 U/ml recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) (both from R&D Systems) for 6 days, to obtain monocyte-derived dendritic cells (MoDC), or with 1000U/ml GM-CSF (Peprotech, London, UK) for 7 days, to obtain monocyte-derived macrophages. Cultures were incubated at 37°C in a 5% CO₂ humidified atmosphere and fresh medium was added every 2/3 days. THP-1 cell line was kindly given by Dr. Nuno Santos (CBME, University of Algarve, Faro, Portugal) and was cultured according to ATCC instructions, in RPMI 1640 with L-glutamine (Lonza, Visp, Switzerland), 10% heat-inactivated FBS (Invitrogen, Carlsbad, CA, USA) and 1% PS (Thermo Scientific, Waltham, MA, USA). Differentiation into macrophagic THP-1 (THP-1 MoM) was achieved by culturing cells in 25 ng/ml of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) in complete RPMI for 48h.