Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit *Green Fluorescence*

Protocol summary

1. Prepare cells in growth medium
2. Co-incubate cells with test compounds and DAX-J2™ PON Green working solution at 37^oC for desired period of time
3. Monitor fluorescence intensity at Ex/Em = 490/530 nm (Cutoff=515 nm)

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

1. DAX-J2™ PON Green stock solution (500X):
Add 20 µL of DMSO (Component C) into the vial of DAX-J2™ PON Green (Component A), and mix well. Note: 20 µL of reconstituted DAX-J2™ PON Green stock solution is enough for 1 plate.

Add 10 μL of 500X DMSO reconstituted DAX-J2™ Peroxynitrite Sensor stock solution into 500 μL of Assay Buffer (Component B) and mix well. Note: The working solution is not stable; prepare it as needed before use.

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

1. Add 10 µL/well (96-well plate), or 2.5 µL/well (384-well plate) of DAX-J2™ PON Green working solution in 90 µL (96-well plate) or 22.5 µL (384-well plate) cell culture per well in the cell plate. Note: It is not necessary to wash cells before staining. It’s recommended to stain the cells in full medium.
   
   
2. Co-incubate cells with DAX-J2™ PON Green with test compounds in full medium or in your desired buffer at 37°C for desired period of time, protected from light. For control wells (untreated cells), add the corresponding amount of compound buffer. Note: It’s recommended to stain the cells in full medium. However, if tested compounds are serum sensitive, growth medium and serum factors can be aspirated away before staining. Add 90 µL/well (96-well plate) and 22.5 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 stained in serum-free media. We co-incubated RAW 264.7 macrophage cells with 50 - 200 µM SIN-1 and DAX-J2™ PON Green in full medium at 37°C for 1 hour to induce peroxynitrite. See Figure 1 for details.
   
   
3. Alternatively, stain cells with DAX-J2™ PON Green at 37°C for 1 hour, protected from light. Remove the working solution, then treat cells with test compounds in full medium or in your desired buffer at 37°C for desired period of time.
   
   
4. Monitor the fluorescence increase using microplate reader at Ex/Em = 490/530 nm (cut off = 515 nm) with bottom read mode, or take images using fluorescence microscope with a FITC filter.