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Protocol summary

1. Prepare cells in growth medium
2. Incubate cells with test compounds and Nitrixyte™ Orange working solution at 37^oC for a desired period
3. Add Assay Buffer II
4. Monitor fluorescence intensity (bottom read mode ) at Ex/Em = 540/590 nm (Cutoff = 570 nm) or fluorescence microscope using TRITC filter

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

Add 20 µL of 500X Nitrixyte™ Orange stock solution (Component A) into 10 mL of Assay Buffer I (Component B) and mix well to make Nitrixyte™ Orange working solution. This Nitrixyte™ Orange working solution is stable for at least 2 hours at room temperature. Protect from light. Note: 20 µL of 500X Nitrixyte™ Orange stock solution is enough for one plate.

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™ Orange working solution in the cell plate. Co-incubate cells with test compound and Nitrixyte™ Orange working solution at 37°C for desired period of time, protected from light. Note: DO NOT remove the test compounds. For a NONOate positive control treatment: Cells were incubated with Nitrixyte™ Orange 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. Note: We have used Raw 264.7 cells incubated with 0.5X 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. See Figure 1 for details.
   
   
3. Remove solution in each well.
   
   
4. 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.
   
   
5. Monitor the fluorescence increase using microplate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm) with bottom read mode, or take images using fluorescence microscope with a TRITC filter.