Immunofluorescence Staining of NF-κB p65

Human monocytic THP-1 cells were cultured on 20-mm diameter glass coverslips in 12-well plates. Cells were pretreated with curcumin (6.25–25 μM) for 1 h and subsequently treated with PMA (100 nM) or vehicle control for 48 h. The cells were immunofluorescence-labeled using a cellular NF-κB translocation kit (Beyotime Biotech), according to the manufacturer’s protocol. Briefly, after washing and fixing, the cells were incubated with a blocking solution at 4°C overnight and subsequently with the NF-κB p65 antibody for 2 h. After washing thrice, a rabbit IgG antibody conjugated with Cy3 was added and incubated for 1 h. To stain the nucleus, the cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. The activation of NF-κB p65 was visualized with an inverted fluorescence microscope (Olympus DP70) at excitation wavelengths of 350 and 540 nm for DAPI and Cy3, respectively. The red and blue images were overlaid to create a two-color image, in which purple fluorescence indicated the areas of colocalization.