2.6. Cytokine Assays

RAW264.7 cells were pre-incubated at 1 × 10⁶ cells·well⁻¹ in a 12-well plate (NEST Biotechnology Co., LTD., Wuxi, China) for 4 h at 37 °C in a 5% CO₂ and then were pretreated with 10 μmol PDTC (inhibitor of NF-κB) before being incubated with various concentrations of EPA (0–3.0 μmol) at 37 °C for 24 h. Levels of IL-1β, IL-6, TNF-α and IFN-γ in the culture supernatants were evaluated by using commercial ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. The absorbance was measured in an ELISA reader at 450 nm. The concentrations of cytokines were calculated according to the standard curve using each of the recombinant cytokines in the ELISA kits.