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.

Figure 1. Jurkat cells were stained with DAF-2DA dye (Kit 9155), washed, and then treated with DMSO control (left histogram) or 1 mM DEA NONOate, a nitric oxide donor (middle histogram). Cells were read on the FL1 channel of a flow cytometer. The median fluorescence intensity (MFI) of DEA NONOate treated cells was 3.5-fold higher than for the control cells. Data also overlaid in a single plot (right, black: Negative; right, red: Positive). Data courtesy of Dr. Kristi Strandberg, ICT, 227:76.
Figure 2. Jurkat suspension cells were stained with DAF-2DA dye, washed, and then treated with DMSO control (Panels A-C) or DEA NONOate (Panels D-F), a nitric oxide donor. Cells treated with DEA NONOate showed an increased level of green fluorescence relative to the untreated cells (Panels A v and D). Microscope images were obtained using a Nikon Eclipse 90i microscope with a Hamamatsu Flash 4.0 camera. Data courtesy of Dr. Kristi Strandberg (ICT 228:57-58).
Figure 3. Jurkat suspension cells were stained with DAF-2DA dye for 1 hour, and then washed. Cells were then treated with DMSO as a negative control or 1 mM DEA NONOate, a nitric oxide donor, for 30 minutes at 37°C. Cells were analyzed using a fluorescence plate reader using an excitation at 488 nm and emission at 515 nm. Data courtesy of Dr. Strandberg, ICT, 227:39-42.
Figure 1. Jurkat cells were stained with DAF-2DA dye (Kit 9155), washed, and then treated with DMSO control (left histogram) or 1 mM DEA NONOate, a nitric oxide donor (middle histogram). Cells were read on the FL1 channel of a flow cytometer. The median fluorescence intensity (MFI) of DEA NONOate treated cells was 3.5-fold higher than for the control cells. Data also overlaid in a single plot (right, black: Negative; right, red: Positive). Data courtesy of Dr. Kristi Strandberg, ICT, 227:76.
Figure 1. Jurkat cells were stained with DAF-2DA dye (Kit 9155), washed, and then treated with DMSO control (left histogram) or 1 mM DEA NONOate, a nitric oxide donor (middle histogram). Cells were read on the FL1 channel of a flow cytometer. The median fluorescence intensity (MFI) of DEA NONOate treated cells was 3.5-fold higher than for the control cells. Data also overlaid in a single plot (right, black: Negative; right, red: Positive). Data courtesy of Dr. Kristi Strandberg, ICT, 227:76.
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Product Manual
Safety Data Sheet

SKU: 9155

Size: 50 - 100 Tests
Price:
$223.00
Ships: 1-2 business days
  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

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