Nitric oxide (NO) plays an important role in neurotransmission, vascular regulation, immune response and apoptosis. NO has been established as one of the key regulators in cardiovascular, nervous, and immune systems. NO plays a pivotal role in numerous processes: in central nervous system, NO participates in cell communication and information storage; in vascular endothelium, NO is involved with regulation of vascular function. Finally, NO is produced by immune cells, including macrophages, as a part of body's defense mechanism during immunological responses and oxidative stress conditions. However, chronic overproduction of NO is one of the fundamental causes underlying disorders including neurodegenerative diseases and pathophysiology of blood vessels. Assay Genie's Nitric Oxide Cell-Based HTS Assay Kit utilizes a dye that reacts with intracellular NO to produce a highly-Fluorescent Triazole Product (Ex/Em= 488/532 nm). The kit includes Diphenyleneiodonium (DPI), a potent inhibitor of nitric oxide cellular production, which has been widely used to inhibit NO synthesis in order to evaluate NO function in different systems and diseases. This kit provides a simple, standardized and HTS method to quantitate the amount of NO in cell-based assays. This Assay Kit is user-friendly, sensitive and can detect the NO Fluorescent product as low as 5 pmol in a 96-well plate

Figure 1: (a) Standard curve of Fluorescent Triazole product (pmol). Standard 0 reading was subtracted from all readings. (b) Macrophage cells (J774A.1) were activated with LPS (200 ng/ml) and γ-IFN (100 ng/ml) for 24 hrs (orange & green bars respectively). The following day, a subset of wells containing activated and non-activated cells were treated with NO inhibitor (DPI 4 µM, blue & green) or vehicle control (0.5% DMSO, grey & orange respectively) for 1 h. After media removal, cells were stained with the NO Staining Dye for 1 h at 37°C, and measurements were taken using spectrophotometer (Ex/Em = 488/532 nm). Fluorescent measurement demonstrated that activated-macrophages produce higher NO levels, that is inhibited by DPI (green bar) to level of non-activated cells. Fluorescent ˆ† RFU was converted to pmol of NO product using equation provided. Note: Background control (CC, in this case), was subtracted from ˆ†RFU reading.  LPS and γ-IFN reagents are not included in kit.