Human granulocyte macrophage colony stimulating factor gm csf

Human monocytes, macrophages and dendritic cells were cultured in RPMI1640‐Glutamax medium supplemented with 1% penicillin/streptomycin 10% FCS at 37°C in 5% CO₂ atmosphere. Buffy coats from healthy donors were obtained freshly from DRK‐Blutspendedienst. Orangu assay was purchased from Cell guidance systems. Human FcR Blocking Reagent, human CD14 MicroBeads, human granulocyte macrophage colony‐stimulating factor (GM‐CSF), macrophage colony‐stimulating factor (M‐CSF), bovine serum albumin (BSA), IL‐4 and all antibodies for surface staining were purchased from Miltenyi Biotec. Cytometric bead array was purchased from BD Biosciences. ELISA for CCL18 and CCL17 was purchased from BosterBio and BioLegend, respectively. Accutase^® solution and Biocoll were purchased from Merck. EDTA was purchased from Sigma‐Aldrich. Silvestrol (purchased from the Sarawak Biodiversity Centre, Kuching, Borneo at a purity of >99%) was dissolved in DMSO and further diluted in medium (c_stock = 6 mmol/L, maximal DMSO concentration during experiments 0.000083% v/v).

PBMCs were isolated by Pancoll density gradient centrifugation (PANbiotech, Aidenbach, Deutschland). CD14⁺ monocytes were isolated by magnetic labeling followed by negative magnetic selection (Pan Monocyte Isolation kit; Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the obtained monocyte fraction was verified by immunostaining. Monocytes were then cultured at 37 °C in AIMV-Albumax medium (Thermofisher Scientific, Walltam, Massachusetts, USA) at the concentration of 1 × 10⁶ cells/mL, supplemented with human Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) (1000 UI/mL; Miltenyi Biotec, Bergisch Gladbach, Germany) and human Interleukin 4 (IL-4) (400 UI/mL; Miltenyi Biotec) (=control condition) or GM-CSF, IL-4 and human HGF (=HGF condition) (20 ng/mL; Miltenyi Biotec). On day 3, the medium was changed, and fresh cytokines were added at the same concentrations. On day 6, culture supernatants and obtained cells were collected. Cytokine dosage and cellular immunostaining were then performed (see corresponding paragraphs). In 7 independent experiments, to provide a maturation signal to DCs, Lipopolysaccharide (LPS) (1 μg/mL; Sigma–Aldrich, Saint-Louis, MI, USA) or control PBS was added to the culture. After 24 h, culture supernatants and cells were collected.