Nitric Oxide Synthase Assay

1. Prepare samples and controls in fresh cell culture medium (alternative buffers such as PBS may be used)., 2. Reconstitute Autophagy Probe, Red with 100 µL DMSO., 3. Dilute Autophagy Probe, Red 1:5 by adding 400 µL diH2O, PBS, or fresh cell culture medium., 4. Add diluted Autophagy Probe, Red to each sample at 1:50 spike (e.g. spike at 1:50 by adding 10 µL to 490 µL sample). The ideal staining concentration can vary based on cell line, application, etc., and should be determined by the end user. The recommended sample size is 0.5 mL., 5. Incubate approximately 1 hour., 6. Dilute 10X Cellular Assay Buffer 1:10 with diH2O., 7. Remove media and wash cells 3 times: add 1X Cellular Assay Buffer or fresh cell culture medium and spin cells., 8. Resuspend cell pellet in 1X Cellular Assay Buffer (typically 0.5 mL per sample)., 9. Analyze with a flow cytometer (using a green/yellow laser and appropriate filter). Autophagy Probe, Red excites at 590 nm and emits at 620 nm.

Nitric oxide synthases are a family of enzymes capable of catalyzing the production of nitric oxide (NO). NO is an important molecule that is involved in regulating a variety of cellular processes such as angiogenesis, peristalsis, and the immune response to invading pathogens. Reactive nitrogen species (RNS) are a family of antimicrobial molecules derived from NO and superoxide through the enzymatic activities of nitric oxide synthase (NOS). ICT’s Nitric Oxide Synthase Assay provides a good screening option for assessing the potency of nitrosative stress inhibitor and activator reagents, and will help to determine how oxidative and nitrosative stress modulates varied intracellular pathways. This kit assesses the overall intracellular levels of free nitric oxide and NOS using a Diaminofluorescein-2 Diacetate (DAF-2DA) dye. The kit provides all the essential reagents and an easy to follow protocol to assess changes in intracellular NO and NOS levels using flow cytometry, fluorescence plate reader, and fluorescence microscopy. The DAF-2DA dye quickly penetrates membrane structures and accumulates within the cell. Once inside the cell the diacetate groups on the DAF-2DA reagent are hydrolyzed by cytosolic esterases, allowing for the release and sequestration of DAF-2 inside the cell. Production of nitric oxide converts the non-fluorescent DAF-2 dye to its fluorescent triaole derivative, DAF-2T3-7. Nitric Oxide Synthase Assay requires minimal procedural steps and hands-on time to complete. Samples are stained for 1 hour with DAF-2DA dye. Cells are washed to remove excess dye, and then treated experimentally. Since the unbound reagent is non-fluorescent, subsequent wash steps are not required, thus simplifying the assay procedure. Following treatment, cells are ready for analysis by flow cytometry. Each kit will enable the assessment of up to 50 (0.5 mL) or 100 (0.25 mL) samples. For microscopy usage, Hoechst 33342 is included with the kit to concurrently label nuclei after labeling with DAF-2DA. DAF-2DA optimally excites at 488 nm, and has a peak emission at 515 nm. Hoechst 33342 can be seen using a UV-filter with excitation at 365 nm and emission at 480 nm.