NF-κB Activation Inhibition Assay

BMDMs (3 × 10⁴ cells/well) seeded into 8-well glass chamber plates were incubated overnight in the presence of 30% of L929-conditioned medium. Cells were pretreated with ChondroT or its constituent herbs for 4 h, and were stimulated with 100 ng/mL of RANKL for 30 min. Cells were then fixed with 4% of paraformaldehyde, and permeabilized using 0.1% of triton. A polyclonal anti-NF-κB p65 antibody (Invitrogen, Carlsbad, MA, USA) and an Alexa Fluor 488-conjugated anti-rabbit IgG second antibody (Molecular Probes Invitrogen, MA, USA) were used for the detection of NF-κB p65 protein. Cells were then mounted with anti-fade reagent with DAPI. Bay 11–7082 was used as a positive control of NF-κB inhibitors. Fluorescence images were photographed with a fluorescence microscope (Nikon DS-Ri2 microscope camera, Tokyo, Japan) [26 (link)].