Fetal calf serum (fcs)

Human RMS cell lines, RD (ERMS) and RH30 (ARMS) [10 (link)], both from American Type Culture Collection,
were respectively cultured in Dulbecco’s modified Eagle’s and RPMI medium containing 10% fetal calf serum (Hyclone, Logan UT) and supplemented with glutamine and gentamycin (GIBCO-BRL Gaithersburg, MD). Human umbilical vein endothelial cells, HUVECs (Clonetics, San Diego, California, USA) were cultured in endothelial cell basal medium (EBM-2; Clonetics) supplemented with 2% of fetal calf serum (FCS; Clonetics) and endothelial growth medium (EGM2; Clonetics). Multipotent mesenchymal stromal cells (MSC) from Wharton’s jelly of umbilical cord [11 (link)], were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum (Hyclone) and supplemented with glutamine and gentamycin (GIBCO-BRL). Cells were incubated at 37 °C in 5% CO2. Medium was replaced every 3 days. Cells from passages 5–7 were used for all the experiments. DNA profiling, using the GenePrint 10 System (Promega Corporation, Madison, WI, USA), was carried out to authenticate cells by comparing the DNA profile of our cell cultures with those found in GenBank. MycoFluor™ Mycoplasma Detection Kit Invitrogen™ was used.

In total, 2 × 10³ BECs were seeded in µ-slide angiogenesis chambers (Ibidi, Munich, Germany) on growth factor-reduced matrigel (BD Biosciences, San Jose, CA, USA). Cells were supplemented with 0, 10, 100 or 1000 ng/ml of recombinant CCL20 (R&D Systems) in EGM-2MV containing 5% FCS (Clonetics). Tube formation was imaged with a Zeiss Cell Observer (Zeiss).

FLS were successfully isolated from RA (n = 6), OA (n = 6) and healthy (n = 6) synovial biopsies subjected to a mild proteolytic treatment with 0.05% trypsin and 0.5 mM EDTA in phosphate-buffered saline (PBS) for 10 minutes at 37 °C under gently shaking. Trypsin was then neutralized with fetal calf serum (FCS) (Celbio, Milan, Italy) and cells were plated in culture dishes (3 cm diameter, BD Falcon, BD Biosciences, San Jose, CA, USA) at a concentration of 1–1.5 × 10⁶ per dish in RPMI 1640 medium (Cambrex Bio Science, Milan, Italy) supplemented with 15% FCS, 2 mM glutamine and penicillin–streptomycin (Cambrex Bio Science). Once at confluence (about 3 weeks), the cell monolayers were expanded in culture by splitting 1:2 every 7 days. The cells were considered FLS (type B synoviocytes) if negative by immunostaining with anti-CD68, anti-CD14, anti-CD11b and anti-CD11c antibodies (markers of type A macrophage-like synoviocytes), positive by staining for the enzyme uridine diphosphoglucose dehydrogenase (preferentially expressed by the intimal lining FLS and reflecting their ability to synthesize hyaluronan, an important constituent of synovial fluid)^{3 (link)}, and if they had a spindle-shaped, fibroblast-like morphology ( > 95% cell purity). Cell monolayers were used within the seventh passage in culture.