M csf

Monocytes were isolated from buffy coat obtained from male healthy blood donors at the department of Transfusion Medicine, County Council of Östergötland, in Linköping, Sweden. All the blood donors had given their informed consent according to the local guidelines (University Hospital in Linköping) and the Swedish National Law on ethical review of research involving humans (2003:460: 3–4 §). The buffy coat was mixed with 70 ml NaCl, layered onto Lymphoprep (Axis-Shield, Oslo) and centrifuged at 480 g in room temperature for 40 minutes. The mononuclear cell layer was collected into new tubes and washed twice with PBS-Heparin for 5 min and centrifuged at 220 g in 4 °C. The white blood cells were seeded to T-75 tissue culture flasks with RPMI 1640 medium, supplemented with 1 U/ml penicillin, 10 μg/ml streptomycin and incubated for 1–2 h to allow monocyte adhesion. The non-adherent cells were eliminated by washing 2–3 times using PBS. The adherent monocytes were allowed to differentiate to macrophages with 40 ng/ml of macrophage colony-stimulating factor, M-CSF (Nordic Biosite, Sweden) for 5–7 days. To induce M2 macrophages, the M-CSF differentiated macrophages were stimulated with 20 ng/ml human interleukin-4, IL-4 (Nordic Biosite, Sweden) for 18–24 h.

Human monocytes derived from healthy donors were isolated from fresh buffy coats supplied by the Finnish Red Cross Blood Service. Monocytes were differentiated into macrophages in Macrophage SFM medium (Gibco) as previously described [20] (link) with a modification of using M-CSF (50 ng/ml; Nordic Biosite) as the growth factor. Primary human mast cells were differentiated from CD34⁺ progenitor cells according to a protocol developed in our laboratory [21] (link), with a minor modification of using Iscove's Modified Dulbecco's Medium, supplemented with BIT 9500 Serum Substitute (Stemcell Technologies) as the cell culture medium (Mast Cell Culture Medium or MCCM) [22] (link). Mast cells were grown for at least 8 weeks before the experiments. The LAD2 mast cell line was originally established from a patient with mast cell sarcoma, and kindly provided by Dr. Dean Metcalfe (NIH, USA) [23] (link). The cells share many characteristics with primary human mast cells and have been commonly used in mast cell research. The LAD2 cells were cultured under serum-free conditions in IMDM containing BIT 9500 Serum Substitute, L-glutamine (2 mM), 2-mercaptoethanol (0.1 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and human recombinant SCF (100 ng/ml) [21] (link).

Immortalized human hepatocytes (IHH) (Samanez et al. 2012) were grown in William's E medium (Gibco/Life Technologies, Carlsbad, CA), 10% fetal bovine serum (FBS), 2 mmol/L l‐glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin, 100 nmol/L (20 mU/mL) insulin and 50 nmol/L dexamethasone (Sigma‐Aldrich, St. Louis, MO), on CellBIND^® plates (Corning, Corning, NY). Human monocytes derived from healthy donors were isolated from fresh buffy coats supplied by the Finnish Red Cross Blood Service. Monocytes were differentiated into macrophages in Macrophage SFM medium (Gibco/Life Technologies, Grand Island, NY) using macrophage colony‐stimulating factor (M‐CSF; 50 ng/mL; Nordic Biosite, Täby, Sweden).