Granulocyte macrophage colony stimulating factor gm csf

Macrophages and MGCs were obtained in vitro, as previously described [69 (link)]. Briefly, human mononuclear cells were obtained from platelet/leukocyte concentrates from healthy donors (convention with Etablissement Français du Sang Aquitaine-Limousin, Bordeaux, France) by centrifugation on a Ficoll gradient (Ficoll-Paque PLUS, GE Healthcare Life Sciences) and were suspended in complete medium Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum (Perbio Science, Cramlington, UK), 2 mM L-glutamine (Invitrogen, Cergy Pontoise, France), 100 IU/mL penicillin (Invitrogen), and 100 μg/mL streptomycin (Invitrogen). Cells were seeded in Primaria® 96-well tissue culture plates (VWR, Strasbourg, France) at 2.5 × 10⁵ cells per well in 100 μL of culture medium. After 1 hour, nonadherent cells were removed by five washings; wells were then added with medium supplemented with granulocyte macrophage colony-stimulating factor (GM-CSF) (100 ng/mL) (Immunotools, Friesoythe, Germany) and the culture was maintained for two weeks. MGCs were generated by IL-4-dependent macrophage fusion: medium supplemented with GM-CSF (100 ng/mL) and IL-4 (100 ng/mL) (Immunotools) was added at day 7, and the culture was maintained for 15 additional days.

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy volunteers (Sanquin, Amsterdam, The Netherlands) by centrifugation on a Ficoll gradient. First, blood was gently mixed with PBS containing 1% citrate and carefully layered on top of Ficoll. After 30 min centrifugation at 700 × g, the interphase containing monocytes and lymphocytes was collected. Next, monocytes and lymphocytes were washed with PBS/Citrate at 400 × g for 10 min and the pellet was resuspended in complete RPMI. To isolate monocytes, PBMCs were carefully added on top of a Percoll layer (GE Healthcare, Chicago, U.S.) at a concentration of 10 × 10⁶ cells/mL and centrifuged at 400 × g for 40 min. Again the interphase was collected and tubes were filled with PBS/Citrate prior to a 10 min centrifugation at 400 × g. After washing three times with PBS/Citrate, pellets were resuspended in complete RPMI. MoDC were generated by culturing monocytes for 5–7 days at a concentration of 1.25^*10⁶/mL in complete RPMI (Lonza, Basel, Switzerland) containing 500 μ/mL IL-4 (ImmunoTools, Friesoythe, Germany) and 800 μ/mL Granulocyte Macrophage Colony stimulating Factor (GM-CSF) (ImmunoTools) using T75 flasks (Greiner, Kremsmünster, Austria).

Epigallocatechin gallat (EGCG), Stattic and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, USA). IFNγ was from BioVision (Milpitas, USA). IL-4 was from Peprotech (Hamburg, Germany). IL-27 was obtained from Biolegend (Koblenz, Germany), IL-27 neutralizing antibody was from Invitrogen (Carlsbad, USA), and the IgG2a istotype control was from BioXCell (West Lebanon, USA). Macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were from ImmunoTools (Friesoythe, Germany). All reagents were dissolved according to the manufacturer's instructions.