Cell Meter™ Intracellular Fluorimetric Hydrogen Peroxide Assay Kit *Blue Fluorescence Optimized for Flow Cytometry*

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 flow cytometer Pacific Blue Channel (Ex/Em = 405/450 nm)

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

1. OxiVision™ Blue Peroxide Sensor stock solution:
Add 100 µL of DMSO (Component B) into the vial of OxiVision™ Blue peroxide sensor (Component A), and mix them well. Note: 1 µL of reconstituted OxiVision™ Blue peroxide sensor stock solution is enough for 0.5 mL cells. The stock solution should be used promptly. Keep from light.

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

1. Stain cells with OxiVision™ Blue Peroxide Sensor stock solution in full medium or in your desired buffer at 37°C for 20 - 30 minutes, protected from light.
   
   
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’s 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. Resuspend cells in 1X Hank’s salt solution and 20 mM Hepes buffer (HHBS) or the buffer of your choice after aspiration. Alternatively, cells can be treated in serum-free media. Note: 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. Monitor the fluorescence intensity at Pacific Blue channel (Ex/Em=405/450 nm) using a flow cytometer. Gate on the cells of interest, excluding debris.