Cell Meter™ Fluorimetric Intracellular Total ROS Activity Assay Kit*Optimized for Flow Cytometry*

Protocol summary

1. Prepare cells at the density of 0.5 - 1 × 10⁶ cells/mL
2. Add 1 µL 500X Amplite™ ROS Green into 0.5 mL cell suspension
3. Stain the cells at 37 ºC for 1 hour
4. Treat the cells to induce ROS
5. Analyze cells using flow cytometer with FL1 channel (Ex/Em = 490/520 nm)

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

1. Amplite™ ROS Green stock solution (500X):
Add 100 µL of DMSO (Component C) into the vial of Amplite™ ROS Green (Component A) and mix well to make 500X Amplite™ ROS Green stock solution. Protect from light.  Note: For storage, seal tubes tightly.

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

1. For each sample, prepare cells in 0.5 mL Assay Buffer (Component B) or buffer of your choice at a density of 5×10⁵ to 1×10⁶ cells/mL. Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for ROS induction
   
   
2. Add 1 µL of 500X Amplite™ ROS Green stock solution into 0.5 mL cell suspension.
   
   
3. Incubate at 37 ºC for 1 hour. Note: For adherent cells, gently lift the cells with 0.5 mM EDTA to keep the cells intact, and wash the cells once with serum-containing media prior to incubation with Amplite™ ROS Green. The appropriate incubation time depends on the individual cell type and test compound used. Optimize the incubation time for each experiment.
   
   
4. Treat cells by adding 50 µL of 11X test compounds in the desired buffer (such as PBS or HBSS). For control wells (untreated cells), add the corresponding amount of buffer. 
   
   
5. Incubate the cells at 37 ºC for a desired period of time to induce ROS, protected from light. Note: We treated Jurkat cells with 100 µM TBHP (tert-Butyl hydroperoxide) at 37 ºC for 30 minutes to induce ROS. See Figure 1 for details.
   
   
6. Monitor the fluorescence intensity using a flow cytometer with FL1 channel (Ex/Em = 490/520 nm). Gate on the cells of interest, excluding debris.