Nitric Oxide (NO) is an anti-bacterial and anti-viral effector of the innate immune system produced by activated immune cells such as monocytes, macrophages, neutrophils and natural killer (NK) cells. Microbial cell wall components and cytokines released from host cells can stimulate the production of inducible NO synthase (iNOS), which in turn generates NO.

Using the iNOS activity assay, compounds can be screened for their ability to inhibit intracellular iNOS activity.  Mouse macrophage cells from the cell line RAW 264.7 are stimulated with LPS and gamma-Interferon (IFN-g) to induce iNOS production. After stimulation, cells are incubated with a reaction mixture containing cell permeable fluorimetric NO indicator and the test article. The resulting fluorescence intensity is proportional to NOS activity. 

Figure: iNOS Activity Assay. RAW 264.7 cells were incubated with LPS and INF-g to induce iNOS production. Following the incubation, media was replaced with vehicle or DPI followed by iNOS reaction mix (containing arginine substrate and DAF-2). Controls included a free sample (no cells), a DAF-2 free sample (no dye) and a non-induced cell sample (non-induced cells). Plates were incubated at room temperature and fluorescence was determined. Mean relative fluorescence units (RFU) are shown. Error bars represent standard deviations.