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 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. Results can be analyzed using a flow cytometer, fluorescence plate reader, or fluorescence microscope.
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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.
Analysis via Flow Cytometry
Jurkat cells were stained with ICT’s DAF-2DA dye (Kit #9155) for 1 hour, washed, and then mock-treated with DMSO to create a negative control (left histogram) or 1 mM DEA NONOate, a nitric oxide donor (middle histogram), for 30 minutes at 37°C. Cells were read on the FL1 channel of an Accuri C6 flow cytometer. The median fluorescence intensity (MFI) of stained cells in the negative control was 324,817 in FL1-A (left : Negative), whereas the treated population had a value of 1,190,365 (middle: Positive), which is a increase of more than 3.5-fold. The effect of DEA NONOate on intracellular NOS activity is easily visible when the samples are overlaid in a single plot (right, black: Negative; right, red: Positive). Data courtesy of Dr. Kristi Strandberg, ICT, 227:76.
Fluorescence microscopy and fluorescent plate reader data can be found in the Nitric Oxide Synthase Assay Manual.
• Excitation/Emission: 488 nm / 515 nm
• Method of Analysis: Flow cytometer, fluorescence plate reader, fluorescence microscopy
• Types of Samples: Cell culture
• Storage: 2-8°C. Protect DAF-2DA dye from light.
• Shipping: Ships overnight (domestic), International Priority Shipping
Kit 9155: 50-100 tests
• Diaminofluorescein-2 Diacetate (DAF-2DA), #6697
• Hoechst 33342, #639
• 10X Assay Buffer, 60 mL, part #685