Cell Meter™ Fluorimetric Intracellular Nitric Oxide (NO) Activity Assay Kit *Red Fluorescence Optimized for Flow Cytometry*

1. Prepare cells (0.5 - 1×10⁶ cells/mL)
2. Add 1 µL 500X Nitrixyte™ Red
3. Incubate cells with test compounds and Nitrixyte™ Red at 37 ºC for desired period of time

4. Analyze cells with a flow cytometer using 610/20 nm filter

Important
Thaw all the components at room temperature before starting the experiment.

For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Add 200 uL of ddH2O into the vial of NONOate Positive Control (Component B) to make 50 mM NONOacte Positive Control treatment stock solution.

Dilute 50 mM NONOate Positive Control treatment stock solution with Assay Buffer (Component C) to make 1-2 mM NONOate positive control working solution.

1. For each sample, prepare cells in 0.5 mL warm medium or buffer of your choice at a density of 5×10⁵ to 1×10⁶ cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for NO induction.

2. Add 1 µL of 500X Nitrixyte™ Red (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™ Red.

3. Incubate cells with test compounds and Nitrixyte™ Red at 37 ºC 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 Nitrixyte™ Red working solution, 20 µg/mL of lipopolysaccharide (LPS) and 1 mM L-Arginine (L-Arg) in cell culture medium at 37 ºC for 16 hours.

4. Spin down cells that have pre-incubated with Nitrixyte™ Red for 30 minutes. Resuspend cells with 1 mM DEA NONOate positive control working solution, and incubate at 37 ºC for another 30 minutes. See Figure 1 for details.

5. Monitor the fluorescence intensity using the 610/20 nm filter in a flow cytometer. Gate on the cells of interest, excluding debris.