2.7. Measurement of cytokine and nitric oxide production from macrophages

RAW264 cells were plated in 24-well culture plates at a density of 2 × 10⁵ cells/cm², and cultured for 24 h. The cells were washed using PBS, and were treated using magnetic lipoplexes (weight ratio of magnetic cationic liposomes:pDNA = 10:1, for each 1 µg of pDNA) for 10 min under a magnetic field. Then, the medium was replaced using fresh RPMI-1640 medium with or without lipopolysaccharide (LPS) (100 ng/mL), and incubated at 37 °C in 5% CO₂/95% air. At 24 h after treatment with the magnetic lipoplexes, the supernatants were collected. The concentrations of IL-6, IL-10, IL-12, and TNF-α in the supernatants were measured using commercial ELISA kits (PeproTech Inc., Rocky Hill, NJ, USA). Nitric oxide (NO) concentrations in the supernatants were measured using a Griess assay, as previously reported (Kono et al., 2016).