4.3. Culture and Stimulation of Isolated Monocytes and THP-1

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.