2.2. Cell Culture and Cell Treatments

THP-1 cells were purchased from Sigma-Aldrich, NZ (Cat. # 88081201) and cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in 5% CO₂. THP-1 cells, at a density of 0.5 × 10⁶ cells / well in 6-well plates, were differentiated into macrophages by treatment with 50 nM PMA for 72 h. Differentiated THP-1 cells were then treated with curcumin and CGA for 1 h, both individually and in combination, at a variety of concentrations (1–25 µM) as indicated in the figure legends. The cells, in the presence of bioactive compounds, were then incubated with LPS (100 ng/mL) for another 4 h to induce an inflammatory response. Cells were pre-treated with curcumin and CGA to ensure full uptake and equilibration prior to LPS stimulation, helping to rule out effects, due to kinetic differences in their absorption versus LPS action. The cell culture supernatants were collected and stored at −80 °C until used for TNF-α quantification by ELISA. The remaining cell monolayer was immediately used to assess cytotoxicity via MTT assay or extracted for subsequent qRT-PCR analysis.