Thp 1 cell line

The THP-1 cell line (ECACC, Salisbury, UK) was an immortalized, human monocytic leukemia derived cell line grown in RPMI 1640 medium (Sigma Aldrich-MERCK) supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO, USA), 2 mMol/L-glutamine (Sigma–Aldrich–MERCK, St. Louis, MO, USA), 100 units/mL penicillin, and 0.1 mg/mL streptomycin (Sigma–Aldrich–MERCK, St. Louis, MO, USA) and kept at 37 °C, 5% CO₂ in a humidified tissue culture incubator.

Isolated human monocytes were allowed to rest for 24 h prior to stimulation in RPMI 1640 Medium (Gibco, ThermoFisher, Paisley, UK) supplemented with 1% L-glutamine (Gibco), 1% penicillin/streptomycin (Gibco) and 5% de-complemented human serum.
THP-1 cell line (Sigma–Aldrich, St-Louis, MO, USA), a human monocytic cell line derived from monocytic leukemia patients, was cultured in RPMI 1640 supplemented with L-glutamine, 10% fetal calf serum (FCS; VWR International Ltd, Lutterworth, UK) and 1% penicillin and streptomycin (PenStrep) at 37 °C with 5% CO₂.
THP-1 or monocytes were seeded into 24-well plates (6 × 10⁵cells/mL) in RPMI 1640 and were left untreated or were treated with 20 ng/mL interleukin-4 (IL-4; Fischer Scientific, Loughborough, UK), 20 ng/mL interleukin-10 (IL-10; PeproTech, London, UK) or 100 ng/mL lipopolysaccharide (LPS; Sigma–Aldrich) for 24 h or in some experiments for the time of 48 h. Cells were washed following stimulation to ensure serum was removed.

The human pro-monocytic THP-1 cell line was purchased from Sigma-Aldrich (St. Louis, MO, USA). The cells were grown at 37 °C in suspension in RPMI 1640 medium GlutaMAX™ (Fisher Scientific; Madrid, Spain) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. THP-1 cells were plated at a density of 3 × 10⁵ in 24-well culture plates and differentiated to macrophages through a first incubation with 100 nM phorbol 12-myristate13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. The PMA-containing media was discarded and replaced with fresh media without PMA for a further 24 h.
The human epithelial cell line BICR-18 (from larynx squamous cell carcinoma) and CAPAN-2 (from pancreatic ductal adenocarcinoma) were obtained from American Type Cell Collection (ATCC, Manassas, VA, USA) and maintained at 37 °C in DMEM GlutaMAX™ (Fisher Scientific; Madrid, Spain) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cultures were split every 3 days by trypsinization with 0.1% trypsin in Ca²⁺/Mg²⁺-free phosphate-buffered saline (PBS) containing 0.9 mM ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich; St. Louis, MO, USA).
All cells were cultured at 37 °C in humidified atmosphere of 95% air and 5% CO₂.