2.9. LPS Stimulation with THP-1 Monocyte-Derived Macrophages

THP-1 cells (human acute monocytic leukaemia cells) were purchased from the European Collection of Authentic Cell Culture (ECACC) and maintained at cell density of 2–9 × 10⁵ cells/mL in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Loughborough, UK) supplemented with 10% heat-inactivated foetal bovine serum (FBS) (Thermo Fisher Scientific, Loughborough, UK) and 2 mM L-glutamine (Thermo Fisher Scientific, Loughborough, UK) in a humidified incubator with 5% CO₂ at 37 °C [22]. A previously described method of LPS-induced inflammation using THP-1 monocyte-derived macrophages was employed to assess the anti-inflammatory activity of the peptides [19]. THP-1 cells were seeded at a density of 2.5 × 10⁵ cells/well in a 24-well plate (Thermo Fisher Scientific, Loughborough, UK) and differentiated to THP-1 monocyte-derived macrophages by culturing under standard cell culture conditions (37 °C/5% CO₂) in the presence of 160 nM phorbol 12-myristate 13-acetate (PMA) (Merck, Gillingham, UK) for 72 h. The cell supernatant was replaced with 1 mL of fresh media and incubated for a further 24 h prior to experimentation. Immediately prior to cell stimulation, cell supernatants were replaced with 500 μL of fresh RPMI (+10% FBS, 2 mM L-glutamine). The cells were incubated with peptide (concentrations indicated in figures) and/or 100 ng/mL Pseudomonas aeruginosa LPS (Serotype 10, Source strain ATCC 27316) (Merck, Gillingham, UK) for 16 h under standard cell culture conditions. The cell supernatants were collected for cytokine quantification.