Cell Meter™ Fluorimetric Intracellular Nitric Oxide (NO) Activity Assay Kit *NIR Fluorescence Optimized for Microplate Reader*

Protocol summary

1. Prepare cells in growth medium
2. Incubate cells with test compounds and Nitrixyte™ NIR working solution
3. Add Assay Buffer II
4. Monitor fluorescence intensity at Ex/Em = 650/680 nm

Important notes
Thaw all the kit component at room temperature before use.

Add 20 µL of Nitrixyte™ NIR stock solution (Component A) into 10 mL of Assay Buffer I (Component B) and mix well. The working solution is stable for at least 2 hours at room temperature. Note: 20 µL of Nitrixyte™ NIR stock solution is enough for one plate. Keep from light.

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

1. To stimulate endogenous NO, treat cells with 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X test compounds (384-well plate) in cell culture medium or your desired buffer (such as PBS or HHBS). For control wells (untreated cells), add the corresponding amount of medium or compound buffer. Note: It is not necessary to wash cells before adding compound. However, if tested compounds are serum sensitive, growth medium and serum factors can be aspirated away before adding compounds. Add 90 µL/well (96-well plate) and 20 µL/well (384-well plate) of 1X Hank’s salt solution and 20 mM Hepes buffer (HHBS) or the buffer of your choice after aspiration. Alternatively, cells can be grown in serum-free media.
   
   
2. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Nitrixyte™ NIR working solution in the cell plate. Co-incubate cells with test compound and Nitrixyte™ NIR working solution at 37°C for desired period of time, protected from light. Note: DO NOT remove the test compounds. Note: For a NONOate positive control treatment: Cells were incubated with Nitrixyte™ NIR working solution at 37°C for 30 minutes. The working solution was removed and cells were further incubated with 1 mM DEA/NONOate at 37°C for 30 minutes to generate nitric oxide. See Figure 1 for details. We have used Raw 264.7 cells incubated with 0.5X Nitrixyte™ NIR, 20 µg/mL of lipopolysaccharide (LPS) and 1 mM L-Arginine (L-Arg) in cell culture medium at 37°C for 16 hours. See Figure 2 for details.
   
   
3. Remove solution in each well. Add Assay Buffer II (Component C), 100 µL/well for a 96-well plate or 25 µL/well for a 384-well plate. Note: DO NOT wash cells before adding Assay Buffer II.
   
   
4. Monitor the fluorescence increase using microplate reader at Ex/Em = 650/680 nm (cut off = 665 nm) with bottom read mode, or take images using fluorescence microscope with a Cy5® filter.