Nitric Oxide Synthase Assay
- 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.