Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Green Fluorescence*

Protocol summary for Solution Assay

1. Prepare and add Amplite™ Green Peroxide Sensor working solution (50 µL)
2. Add H₂O₂ standards or test samples (50 µL)
3. Incubate at room temperature for 15 - 60 minutes
4. Read fluorescence intensity at Ex/Em = 490/525 nm (Cutoff = 515 nm)

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

Protocol summary for Live Cell Assay

1. Prepare cells in growth medium
2. Stain cells with Amplite™ Green Peroxide Sensor working solution and incubate for your desired period of time
3. Treat cells with test compounds
4. Monitor fluorescence intensity at Ex/Em = 490/525 nm (Cutoff = 515 nm) with bottom read mode

Important notes
Amplite™ Green Peroxide Sensor can be loaded passively into living cells and report the micromolar changes in intracellular H₂O₂ concentrations. The following is a suggested microscope imaging protocol that can be modified to meet specific research needs.

1. Amplite™ Green Peroxide Sensor stock solution (250X):
Add 50 µL of DMSO (Component D) into the vial of Amplite™ Green Peroxide Sensor (Component A) to make 250X Amplite™ Green Peroxide Sensor stock solution. Protect from light.

2. H₂O₂ standard solution (20 mM):
Add 22.7 µL of 3% H₂O₂ (0.88 M, Component B) into 977 µL of Assay Buffer (Component C) to make 20 mM H₂O₂ standard solution. Note: The diluted H₂O₂ standard solution is not stable. The unused portion should be discarded.

Add 20 μL of 250X Amplite™ Green Peroxide Sensor stock solution into 5 mL of Assay Buffer (Component C) to make Amplite™ Green Peroxide Sensor working solution.

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

Run H₂O₂ assay in supernatants reaction:

Table 1. Layout of H₂O₂ standards and test samples in a solid black 96-well microplate. HS= H₂O₂ Standards (HS1 - HS7, 300 to 0.3 µM); BL=Blank Control; TS=Test Samples

Table 2. Reagent composition for each well.

1. Prepare H₂O₂ standards (HS), 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 Amplite™ Green Peroxide Sensor working solution to each well of H₂O₂ standard, blank control, and test samples to make the total H₂O₂ assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Amplite™ Green Peroxide Sensor working solution into each well instead, for a total volume of 50 µL/well.
   
   
3. Incubate the reaction at room temperature for 15 to 30 minutes, protected from light.
   
   
4. Monitor the fluorescence increase with a fluorescence plate reader at Excitation = 490 ± 10, Emission = 520 ± 10 nm (optimal Ex/Em = 490/525 nm, Cutoff = 515 nm).

Run H₂O₂ assay in live cells:

1. Activate the cells as desired.
   
   
2. Wash the cells with PBS buffer, incubated the cells with 100 µL/well Amplite™ Green Peroxide Sensor working solution for 5 to 60 minutes or your desired time. For a 384-well plate, add 25 µL/well of Amplite™ Green Peroxide Sensor working solution. Note: For a kinetic measurement, cells can be stained before adding the treatment.
   
   
3. Monitor the fluorescence increase with a fluorescence plate reader (bottom read mode) at Ex/Em = 490/525 nm (Cutoff = 515 nm) Or image the fluorescence change with a fluorescence microscope using FITC channel.