RAW 264.7 mouse macrophage cells were cultured in DMEM supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 lg/mL) and held at 378C with 7.5% carbon dioxide in a 96-well flat-bottom tissue culture plate for 12 h. Cells were then treated with LPS (1 lg/mL) alone (positive control) or LPS with various concentrations
of X for 18 h. The cell supernatants were collected at the end of the culture for nitrite, which were used as a measure of NO production [25]. Equal volumes of Griess reagent (100 lL; Sigma-Aldrich) were added to each cell supernatant (100 lL), and the absorbance was measured at 570 nm. The concentration of nitrite (lM) was calculated from a standard curve drawn with a known concentration of sodium nitrite dissolved in DMEM. The results are presented as the mean l SD of 4 replicates of one representative experiment and this experiment has been repeated three times with similar results.
