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

Protocol summary

1. Prepare cells in growth medium
2. Stain cells with OxiVision™ Blue Peroxide Sensor
3. Treat cells with test compounds
4. Monitor fluorescence intensity with DAPI filter or Ex/Em = 405/450 nm

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

1. OxiVision™ Blue Peroxide Sensor stock solution (500X):
Add 40 µL of DMSO (Component C) into the vial of OxiVision™ Blue Peroxide Sensor (Component A) and mix well to make 500X OxiVision™ Blue Peroxide Sensor stock solution.  Note: 20 µL of reconstituted OxiVision™ Blue peroxide sensor stock solution is enough for 1 plate. The stock solution should be used promptly. Protect from light.

Add 10 μL of 500X OxiVision™ Blue Peroxide Sensor stock solution into 500 μL of Assay Buffer (Component B) and mix well to make OxiVision™ Blue Peroxide Sensor working solution. Note: This OxiVision™ Blue Peroxide Sensor 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 OxiVision™ Blue Peroxide Sensor working solution in 90 µL cell culture per well in a 96-well cell plate or 22.5 µL cell culture per well in a 384-well cell plate. Note: It is not necessary to wash cells before staining. It’s recommended to stain the cells in full medium.
   
   
2. Treat cells with test compounds in full medium or in your desired buffer at 37°C for desired period of time. For control samples (untreated cells), add the corresponding amount of compound buffer. Note: It is recommended to treat cells in full medium. However, if tested compounds are serum sensitive, growth medium and serum factors can be aspirated away before treatment. Add 1X Hank’s salt solution and 20 mM Hepes buffer (HHBS) or the buffer of your choice into the cells after aspiration. Alternatively, cells can be treated in serum-free media. We treated Jurkat cells with 100 µM hydrogen peroxide in full medium at 37°C for 90 minutes to induce hydrogen peroxide. See Figure 1 for details.
   
   
3. Wash cells with DPBS 1 - 2 times, and replace with 100 uL/well (for 96-well plate) or 25 uL/well (for 384- well plate) Assay Buffer (Component C).
   
   
4. Take images using fluorescence microscope with a DAPI filter.