Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Red Fluorescence*

Protocol summary

1. Prepare cells in growth medium
2. Add Amplite™ ROS Red working solution (100 µL/well for a 96-well plate or 25 µL/well for a 384-well plate)
3. Incubate the cells at 37°C for 1 hour
4. Treat the cells with test compounds to induce ROS
5. Monitor the fluorescence increase (bottom read mode) at Ex/Em= 520/605 nm (Cutoff = 590 nm) or fluorescence microscope with Ex/Em = 520/605 nm filter set

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

1. Amplite™ ROS Red stock solution (500X):
Add 40 µL of DMSO (Component C) into the vial of Amplite™ ROS Red (Component A) and mix well to make 500X Amplite™ ROS Red stock solution. Protect from light. Note: 20 µL of 500X Amplite™ ROS Red stock solution is enough for 1 plate. Note: Unused portion can be aliquoted and stored at < -20 °C for more than one month if the tubes are sealed tightly and kept from light. Avoid repeated freeze-thaw cycles.

Add 20 µL of 500X Amplite™ ROS Red stock solution into 10 mL of Assay Buffer (Component B) and mix well to make Amplite™ ROS Red working solution. Note: This Amplite™ ROS Red working solution is stable for at least 2 hours at room temperature.

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

1. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Amplite™ ROS Red working solution into the cell plate.
   
   
2. Incubate the cells in a 5% CO₂, 37°C incubator for one hour.
   
   
3. Treat cells with 20 µL of 11X test compounds (96-well plate) or 10 µL of 6X test compounds (384-well plate) in your desired buffer (such as PBS or HHBS). For control wells (untreated cells), add the corresponding amount of compound buffer.
   
   
4. To induce ROS, incubate the cell plate at room temperature or in a 5% CO₂, 37°C incubator for at least 15 minutes or a desired period of time (30 minutes for Hela cells treated with 1 mM H₂O₂).
   
   
5. Monitor the fluorescence increase with a fluorescence microplate reader (bottom read mode) at Ex/Em = 520/605 nm (Cutoff = 590 nm) or observe cells using a fluorescence microscope with Ex/Em = 520/605 nm filter set (Texas Red filter).