NF-κB activation and nuclear translocation assay

RAW264.7 cells (1 × 10⁵ cells/well) seeded in a 6-well plate were treated with CPS or LPS for 6 h. Then, the supernatants were discarded, and the immunofluorescence test was performed according to the instructions from the manufacturer of NF-κB activation nuclear translocation assay Kit (Beyotime, Jiangsu, China). Cells were fixed with the stationary liquid for 15 min and blocked with blocking solution for 1 h at room temperature. The cells were then incubated overnight at 4°C with the primary antibody NF-κB. Following washing, the NF-κB antibody was recycled and further incubated with Cy3-conjugated secondary antibody for 1 h and stained with 4′, 6-diamidino-2-phenylindole (DAPI) for 5 min. Images were captured using a TS2-FL microscope (Nikon, Tokyo, Japan).