Isolation and Differentiation of Fibroblasts and Monocytes

Fibroblasts were isolated as previously described (10 (link)). Fibroblasts were grown in media containing 10% foetal bovine serum (FBS), sodium 0.87 mM orthopyruvate, 0.87x MEM Non-essential amino acids, 1.75 mM glutamine, 87 U/ml penicillin and 87 ug/ml streptomycin. After 3-4 passages and reaching 80-90% confluence, fibroblasts were detached and used for specific experiments. All cells were at the same passage number for all experiments. Human monocytes from healthy blood donors were obtained from blood cones supplied by the National Blood and Transplant Service (ethical approval ERN_16-0191). Monocytes were isolated by negative selection using RosetteSep Human Monocyte Enrichment Cocktail (STEMCELL Technologies). Cells were then differentiated for 7 days in RPMI 1640 (Thermo Fisher Scientific) supplemented with 5% heat-inactivated FBS in presence of recombinant macrophage colony-stimulating factor (M-CSF) (50 ng/ml; PeproTech) and then used for in vitro experiments.