Amplite® Fluorimetric Extracellular Nitric Oxide (NO) Activity Assay Kit

Protocol summary

1. Prepare nitric oxide working solution (50 µL)
2. Add nitric oxide standard or test samples (50 µL)
3. Incubate at room temperature for 30 - 60 minutes
4. Monitor the fluorescence intensity

Important notes
Thaw one of each kit component at room temperature before starting the experiment.

1. DAW-J2 stock solution (200X): 
   Add 25 mL of DMSO into the vial of DAW-J2 (Component A) to make 200X stock solution.
   
   
2. Nitric oxide standard solution (100 mM, not provided):
   We used DEA NONOate (Cayman Chemical, Item No. 82100, CAS#372965-00-9) as the nitric oxide standard. The standard solution of nitric oxide was prepared at the concentration of 100 mM in 0.01N NaOH.

Add 25 µL of DAW-J2 200X stock solution into 5 mL of Assay Buffer (Component B) and mix well. Note: This nitric oxide working solution is enough for 100 assays. The working solution is not stable, use it promptly and avoid direct exposure to light.

Table1: Layout of nitric oxide standards and test samples in a solid black 96-well microplate. SD=Standard, BL=Blank Control, TS=Test Sample.

Table2: Reagent composition for each well.

1. Prepare nitric oxide standards (SD), blank controls (BL) and test samples (TS) into a solid black 96-well microplate according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL per well.
   
   
2. Add 50 µL of nitric oxide working solution to each well of standards, blank controls and test samples to make the total nitric oxide assay volume of 100 µL/well. For a 384-well plate, use 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 30 minutes to 1 hour, protected from light.
   
   
4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 570 nm, Emission = 610 nm (cutoff = 590 nm).