The Effect of Lithium on Inflammation-Associated Genes in Lipopolysaccharide-Activated Raw 264.7 Macrophages

Figure 8

Evaluation of the effects of lithium on nuclear translocation of NF-κB after LPS activation of Raw 264.7 cells. Cells were cultured at a density of 2 × 10⁵ cells/ml on coverslips and treated with 10 mM lithium, 10 mM NaCl, 100 ng/ml ultrapure LPS, and a combination of salts and LPS for 90 min. Cells were then fixed with 3.7% paraformaldehyde and thereafter semipermeabilised with 0.1% Triton-X100 in 1% BSA for 30 min. The nonspecific binding was blocked for an hour with 1% BSA and then anti-NF-κB antibody (1 : 500) was added for an hour, and then the cells were washed 3x with 1x PBS and stained for 30 min with anti-rabbit IgG-FITC (1 : 2000). After 30 min incubation, cells were washed, followed by DNA staining with 25 μg/ml Hoechst for 20 min. Thereafter, coverslips were mounted on glass slides with mounting medium and then NF-kB was analysed and photographed under fluorescence inverted microscope (Nikon Eclipse TS100F, Japan).