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

Protocol summary

1. Prepare cells in growth medium
2. Stain cells with OxiVision™ Green peroxide sensor
3. Treat cells with test compounds
4. Monitor fluorescence intensity with flow cytometer FITC channel (Ex/Em = 490/530 nm)

Important notes
Thaw all the four components at room temperature before use.

1. OxiVision™ Green Peroxide Sensor stock solution:
Add 100 µL of DMSO (Component B) into the vial of OxiVision™ Green Peroxide Sensor (Component A), and mix well. Note: 1 µL of reconstituted OxiVision™ Green Peroxide Sensor stock solution is for 0.5 mL cells. The stock solution should be used promptly. Protect 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™ Green Peroxide Sensor stock solution in full medium or in your desired buffer at 37°C for 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 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. 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. Alternatively, treat cells with tested compounds at 37°C for desired period of time. Remove the treatment solution, then stain cells with OxiVision™ Green Peroxide Sensor stock solution in full medium or in your desired buffer at 37°C for desired period of time.
   

4. Monitor the fluorescence intensity at FITC channel (Ex/Em = 490/530 nm) using a flow cytometer. Gate on the cells of interest, excluding debris.