Cell Meter™ Fluorimetric Intracellular Nitric Oxide (NO) Activity Assay Kit *Orange Fluorescence Optimized for Flow Cytometry*
|Shipping||Standard overnight for United States, inquire for international|
|H-phrase||H303, H313, H333|
|Intended use||Research Use Only (RUO)|
|R-phrase||R20, R21, R22|
|Excitation||488 nm laser|
|Emission||575/26 nm filter|
|Instrument specification(s)||PE channel|
|Component A: 500X Nitrixyte™ Orange||1 vial (100 µL)|
|Component B: NONOate Positive Control||1 vial (lyophilized powder)|
|Component C: Assay Buffer||1 bottle (10 mL)|
AT A GLANCE
- Prepare cells (0.5 - 1 × 106 cells/mL)
- Add 1 µL 500X Nitrixyte™ Orange into 0.5 mL cell suspension
- Incubate cells with test compounds and Nitrixyte™ Orange at 37°C
- Analyze with a flow cytometer
Thaw all the components at room temperature before use.
PREPARATION OF STOCK SOLUTION
1. NONOate Positive Control stock solution (50 mM):
Add 200 µL of ddH2O into the vial of NONOate Positive Control (Component B) to make 50 mM stock solution.
PREPARATION OF WORKING SOLUTION
1. NONOate Positive Control working solution
Dilute the NONOate Positive Control stock solution to a 1 - 2 mM working solution with Assay Buffer (Component C).
For guidelines on cell sample preparation, please visit
SAMPLE EXPERIMENTAL PROTOCOL
- Add 1 µL of 500X Nitrixyte™ Orange (Component A) into 0.5 mL cell suspension. Note: For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact, and wash the cells once with serum-containing media prior to incubation with Nitrixyte™ Orange.
- Incubate cells with test compounds and Nitrixyte™ Orange at 37°C for a desired period of time to generate endogenous or exogenous NO. Note: The appropriate incubation time depends on the individual cell type and test compound used. Optimize the incubation time for each experiment. Note: We have used Raw 264.7 cells incubated with 1X Nitrixyte™ Orange, 20 µg/mL of lipopolysaccharide (LPS) and 1 mM L-Arginine (L-Arg) in cell culture medium at 37°C for 16 hours.
- Spin down cells that have pre-incubated with Nitrixyte™ Orange for 30 minutes. Resuspend cells with 1 mM DEA NONOate positive control working solution, and incubate at 37°C for another 30 minutes. See Figure 1 for details.
- Monitor the fluorescence intensity at the FL2 channel (Ex/Em = 488/590 nm) using a flow cytometer. Gate on the cells of interest, excluding debris.
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