Human granulocyte macrophage colony stimulating factor

REH (#ACC-22), TF-1 (#ACC334) and L929 (#ACC-2) cells were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany), KYM-1 cells were kindly provided by Harald Wajant (University of Wuerzburg). All cell lines were grown RPMI 1640 medium (PAN Biotech, Aidenbach, Germany) supplemented with 10% (v/v) fetal calf serum (Sigma, Steinheim, Germany). TF-1 cells were additionally supplemented with 5 ng/ml human granulocyte–macrophage colony-stimulating factor (Immunotools, Friesoythe, Germany). Antibodies used in the study: TNF #3707, p38 #9212, phospho-p38 #9211, ERK #4695, phospho-ERK #4370 (Cell Signaling, Beverly, MA, USA); tubulin #MS-581: Dunnlab (Asbach, Germany); NFAT5 #PA1-023: Thermo Fisher Scientific (Waltham, MA, USA). Chemicals: MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide): Biomol (Hamburg, Germany); zVAD-fmk (carbobenzoxy-valyl-alanyl-aspartyl-(Omethyl)-fluoromethylketone): Bachem (Bubendorf, Switzerland); BIRB796, PH797804, SB203580, BV6, LCL161 and birinapant: Selleck Chemicals (Houston, TX, USA). Necrostatin-1 (Nec-1): Stress-Marq (Victoria, Canada); Etanercept was obtained from Pfizer (Berlin, Germany).

Human monocyte-derived immature DCs were generated from peripheral blood mononuclear cells (PBMCs) obtained from buffy coat preparations (National Blood Centre, St. James’s Hospital, Dublin) by density gradient centrifugation (Lymphoprep) as we previously described (Supplementary Fig. 1 A) [18 (link), 43 (link)]. Briefly, monocytes were isolated by positive selection using anti-CD14 magnetic microbeads as described by the manufacturer (Miltenyi Biotec) and seeded at a density of 1 × 10⁶ cells/mL in 6-well plates in 3 mL of RPMI-1640 medium containing 10% defined HyClone FBS (Thermo Scientific), 1% penicillin/streptomycin, 1% fungizone, human granulocyte macrophage colony-stimulating factor (50 ng/mL; Immunotools), and human IL-4 (70 ng/mL; Immunotools) in a humidified atmosphere with 5% CO₂ at 37 °C. Cells were fed at day 3 by replacing half the medium made up with fresh cytokines as above. At day 6, CD11c⁺ cells exhibited an immature DC phenotype capable of upregulating cell surface markers following LPS activation.

Human monocyte-derived immature DCs were generated from peripheral blood mononuclear cells (PBMCs) obtained from buffy coat preparations (National Blood Centre, St. James’s Hospital, Dublin) by density gradient centrifugation (Lymphoprep) as described [29 (link), 30 (link)]. Briefly, monocytes were isolated by positive selection using anti-CD14 magnetic microbeads as described by the manufacturer (Miltenyi Biotec) and seeded at a density of 1 × 10⁶ cells/ml in 6-well plates in 3 ml of RPMI-1640 medium containing 10% defined HyClone FBS (Thermo Scientific), 1% penicillin/streptomycin, 1% fungizone, human granulocyte macrophage colony-stimulating factor (50 ng/ml; Immunotools), and human IL-4 (70 ng/ml; Immunotools) in a humidified atmosphere with 5% CO₂ at 37 °C. Cells were fed at day 3 by replacing half the medium made up with fresh cytokines. At day 6, CD11c⁺ cells exhibited an immature DC phenotype, as confirmed by ability to upregulate maturation markers following LPS stimulation.