Nitric Oxide Synthase Assay

Assess the potency of nitrosative stress inhibitor and activator reagents. Measure the overall intracellular levels of free nitric oxide and NOS using a DAF-2DA dye. Analyze on a flow cytometer, fluorescence plate reader, or fluorescence microscope.



SKU: 9155

Size: 50 - 100 Tests
Price:
Sale price$216

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.

Nitric Oxide, NOS
488 nm / 515 nm
Flow cytometry, Fluorescence microscope, fluorescence plate reader
Cell culture
2-8°C
Domestic: Overnight Delivery; International: Priority Shipping
United States
  1. 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.
  2. 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.
  3. 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.
  4. Incubate 1 hour at 37°C.
  5. Wash samples at least once with 1X Assay Buffer to remove excess dye, and then resuspend in 1X Assay Buffer.
  6. Treat cells with test compounds for desired period of time to induce NOS production. Keep cells protected from light.
  7. Analyze with a flow cytometer, fluorescence plate reader, or fluorescence microscope. DAF-2DA excites at 488 nm and emits at 515 nm.
  • Diaminofluorescein-2 Diacetate (DAF-2DA), #6697
  • Hoechst 33342, #639
  • 10X Assay Buffer, 60 mL, part #685
  • Bulk Order Nitric Oxide Synthase Assay

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