Amplite® Fluorimetric Peroxynitrite Quantification Kit *Green Fluorescence*

Important notes
Thaw all the kit components to room temperature before use. 

1. DAX-J2™ PON Green 99 stock solution (500X):
Add 20 µL of DMSO (Component C) into the vial of DAX-J2™ PON Green 99 (Component A) to make 500X stock solution. Note: 20 µL of reconstituted DAX-J2™ PON Green 99 stock solution is enough for 1 plate. Keep from light.

2. Peroxynitrite (ONOO¯ ) stock solution (not provided):
Peroxynitrite stock solution was synthesized according to literature report. Briefly, a mixture of sodium nitrite (0.6 M) and hydrogen peroxide (0.7 M) was acidified with hydrochloric acid (0.6 M), and sodium hydroxide (1.5 M) was added within 1 - 2 seconds to make the solution alkaline. The excess hydrogen peroxide was removed by passing the solution through a short column of manganese dioxide. The extinction coefficient of peroxynitrite solution in 0.1 M NaOH is 1670 M^-1cm^-1 at 302 nm. The ONOO¯ stock solution is not stable; we highly recommend make it fresh to use.

Add 20 μL of 500X DAX-J2™ PON Green 99 stock solution into 10 mL of Assay Buffer (Component B) and mix well. Note: This assay mixture is enough for one 96-well plate. Protect from light.

Table 1. Layout of ONOO¯ standards and test samples in a solid black 96-well microplate. O = ONOO¯ Standards (O1 - O7, 0.313 to 20 µM); BL = Blank Control; TS = Test Samples.

Table 2. Reagent composition for each well.

1. Prepare ONOO¯ standards (O), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
   
   
2. Add 50 µL of working solution to each well of ONOO¯ standard, blank control, and test samples to make the total ONOO¯ assay volume of 100 µL/well. For a 384-well plate, add 25 µL of working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction at room temperature for 5 to 10 minutes, protected from light.
   
   
4. Monitor the fluorescence increase at Ex/Em = 490/530 nm (cutoff at 515 nm) with a fluorescence plate reader.