Measuring Inflammatory Responses in RAW264.7 Cells

RAW264.7 cells (TIB-71, ATCC, Manassas, VA, USA) were obtained from American Type Culture Collection (ATCC). NO production was measured using a previously described method [43 (link),44 (link)]. Briefly, RAW264.7 macrophages were seeded into 96-well plates at a density of 5 × 10⁵ cells/well, and after being incubated with samples for 1 h, the cells were treated with LPS (0.1 µg/mL) for 24 h. Equal volumes of the cell culture supernatant and Griess reagent were mixed, and the absorbance of the mixtures was recorded at 550 nm. The levels of PGE₂, IL-1β and IL-6 cytokines were measured with ELISAs. RAW264.7 cells (2 × 10⁶ cells/well) were cultured in a 6-well plate for 24 h. The cells were activated by LPS (0.1 µg/mL) for 16 h after being pretreated with samples for 1 h, and the concentrations of PGE₂, IL-1β and IL-6 in the supernatants were measured using an ELISA kit (R&D Systems, Minneapolis, MN, USA). The cell viabilities were quantified with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay after 24 h of incubation with the samples [45 (link)].