Amplite® Fluorimetric Hypochlorite (Hypochlorous Acid) Assay Kit

Protocol summary

1. Prepare Hypochlorite working solution (50 µL)
2. Add Hypochlorite standards or test samples (50 µL)
3. Incubate at room temperature for 10 - 30 min
4. Monitor fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important notes
To achieve the best results, it’s strongly recommended to use the black plates. Thaw one vial of each kit component at room temperature before starting the experiment.

1. Oxirite™ Hypochlorite Sensor stock solution (200X):
Add 50 µL of DMSO (Component D) into the vial of Oxirite™ Hypochlorite Sensor (Component A) to make 200X stock solution. Protect from light.

Add 25 µL of 200X Oxirite™ Hypochlorite Sensor stock solution into 5 mL of Assay Buffer (Component B) and mix well to make Hypochlorite working solution. Note: This Hypochlorite working solution is enough for one 96-well plate. It is not stable, use it promptly.

Table 1. Layout of Hypochlorite standards and test samples in a 96-well solid black microplate. H= Hypochlorite Standards (H1 - H7, 0.001 to 100 mM), BL=Blank Control, TS=Test Samples. 

Table 2. Reagent composition for each well.

1. Prepare Hypochlorite standards (H), 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 Hypochlorite working solution to each well of Hypochlorite standard, blank control, and test samples to make the total Hypochlorite assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Hypochlorite working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction at room temperature for 10 - 30 minutes, protected from light.
   
   
4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm).