Inhibition of iNOS activity

The assay was performed using mouse macrophages 915 (RAW 264.7, obtained from ATCC). Cells were cultured in phenol 916 red free RPMI medium supplemented with 10% bovine calf serum, 100 U/mL penicillin G sodium, and 100 µg/mL streptomycin at 37°C in an atmosphere of 5% CO₂ and 95% humidity. Cells were seeded in 96-well plates at 5×10⁴ cells/well and incubated for 24 h. Test compounds diluted in serum-free medium were added to the cells. After 30 min of incubation, LPS (5 µg/mL) was added and the cells were further incubated for 24 h. The concentration of nitric oxide (NO) was determined by measuring the level of nitrite released in the cell culture supernatant using Griess reagent.²⁵ Percent inhibition of nitrite production by the test compound was calculated in comparison to the vehicle control. IC₅₀ values were obtained from dose curves. Parthenolide was used as a positive control.^26,27