Recombinant human il 1

We used 2 PDAC cell lines, BxPC-3 and PANC-1 (American Type Culture Collection, Manassas, VA, USA). An immortalized cell line derived from human pancreatic duct, HPDE, was a kind gift from Dr. M.S. Tsao (Univ. of Toronto, Canada). PDAC cell lines were maintained in RPMI1640 medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Life Technologies) and 1% streptomycin and penicillin (Life Technologies). HPDE was maintained in HuMedia-KG2 (KURABO, Osaka, Japan), in a 5% CO₂ incubator at 37°C. Recombinant human IL-1ß (PEPROTECH, Rocky Hill, NJ, USA) and NS-398 (Sigma-Aldrich, St.Louis, MO, USA) were used at different concentrations (IL-1ß: 500pg/ml, NS-398: 100, 200μM) for 48 hours treatment.

The cytotoxic effect of the V. thapsus was evaluated by colorimetric assay MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma-Aldrich), as previously reported [35 (link)]. For MTT 5 × 10³ cells/well were cultured in 96 wells and after 24 h, a fresh medium containing several concentrations of V. thapsus and H. procumbens was added. Following 24 h and 6 days of incubation, the medium was removed, LPS (1 µg/mL) (O26:B6 E. coli, Sigma-Aldhric) and Il-1β (10 ng/mL) (recombinant human Il-1ß (PeproTech EC, London, UK)) was added for 24 h. After the cells were washed and 200 µL of MTT solution (1 mg/mL in FBS-free medium) was added to each well and incubated for 2 h at 37 °C and 5% CO₂. Following 2 h of incubation, the medium was removed, each well was washed two times using cold PBS, and the formed crystals were melted using 200 μL of DMSO. The absorbance at 570 nm was read using a synergy HT plate reader (BioTek Instruments, Inc., Winooski, VT, United States).

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.