Penicillin streptomycin amphotericin

FTC-133 (Sigma-Aldrich, Italy) and 8505c (Sigma-Aldrich, Italy) human cell lines were used in this analysis. FTC-133 cells were cultured in DMEM: Ham’s F12 (1:1) (Sigma-Aldrich, Italy) supplemented with L-Glutamine 2 mM (Euroclone, Italy), penicillin/streptomycin/amphotericin (PSA) (Euroclone, Italy), and 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich, Italy), while 8505c cells were grown in EMEM (Sigma-Aldrich, Italy) containing 1% of non-essential amino acids, L-Glutamine 2 mM, PSA and 10% FBS. Cells were maintained in a humidified environment at 37 °C and 5% CO₂. The medium was replaced twice a week and cells were split at about 80% to 90% of confluence.

Two different cell lines, mouse leukemic monocyte-macrophage (RAW 264.7, Sigma-Aldrich, Milan, Italy) and human adult chondrocytes (HC, Sigma-Aldrich) were used in this study. RAW 264.7 cells were cultured in EMEM (Sigma-Aldrich) supplemented with 2 mM L-Glutamine (Euroclone, Milan, Italy), 1% Non-Essential Amino Acids (Sigma-Aldrich), 10% Fetal Bovine Serum (FBS, Sigma-Aldrich) and penicillin/streptomycin/amphotericin (PSA) (Euroclone), while HC cells were grown in Chondrocyte Growth Medium (Sigma-Aldrich) containing PSA. Cells were maintained in a humidified environment at 37 °C and 5% CO₂/95% air atmosphere and cultured in T75 flasks. The medium was replaced twice a week and cells were split at about 60–80% of confluence. Treatments with V. thapsus and H. procumbens were performed by adding different concentrations of each extract (50, 100 and 200 µg/mL) to the culture medium for 24 h and 6 days before inducing inflammation in cells with LPS (1 µg/mL) (O26:B6 E. coli, Sigma-Aldhric) and Il-1β (10 ng/mL) (recombinant human Il-1ß (PeproTech EC, London, UK)). Next, the medium was removed and further analyses were carried out.