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. Results can be analyzed using a flow cytometer, fluorescence plate reader, or fluorescence microscope.
- Prepare samples and controls in 0.5 mL 1X Assay Buffer or buffer of choice at a cell density between 3-5 x 105 cells/mL. If samples were cultured in serum-containing medium, wash the samples prior to adding the DAF-2DA dye.
- Prepare DAF-2DA staining solution by adding 56 µL DAF-2DA to 444 µL water. This is sufficient volume to stain 50 x 0.5 mL or 100 x 0.25 mL samples.
- Pre-load samples with DAF-2DA staining solution by adding 10 µL into 490 µL cultured cells, or 5 µL into 245 µL cultured cells.
- Incubate 1 hour at 37°C.
- Wash samples at least once with 1X Assay Buffer to remove excess dye, and then resuspend in 1X Assay Buffer.
- Treat cells with test compounds for desired period of time to induce NOS production. Keep cells protected from light.
- Analyze with a flow cytometer, fluorescence plate reader, or fluorescence microscope. DAF-2DA excites at 488 nm and emits at 515 nm.