ROS determination

1. We seeded a lot of cells in 12 well-plates.

2. After 24 h, cells were washed and stained with 10 μM dihydroethidium (DHE, 37,291, Sigma Aldrich) for 30 min at 37°C, protected from light.

3. Cells were then washed, trypsinized and centrifuged at 2,500 rpm.

4. The pellet was resuspended in PBS with 3% FBS, filtered and analyzed in a BD Facs Canto II flow cytometer (Ex: 488 nm, Em: 675 nm).

or in black plates and fluroskan (if you do not have cytometer)

That's all

Thank you very much for your response, Juan.

I have a few additional questions regarding the positive control (hydrogen peroxide). I have come across different protocols—some suggest adding H₂O₂ after DCFDA incubation, while others indicate that it should be added during the incubation period. Which approach would you recommend for optimal ROS detection?

Additionally, I have seen that Menadione might be a more suitable positive control. Do you have any insights on whether it provides a more reliable ROS induction compared to H₂O₂? My plan is to test both compounds at different concentrations to evaluate their effectiveness.

I appreciate any advice on this. Thanks in advance!

Please read:

https://www.nature.com/articles/s42255-022-00591-z.

You may come to the conclusion that such kits do not provide useful information. As ROS is a misnomer, you should determine what you want to detect. All the species collected under "ROS" have vary different properties. For instance, there are those that claim that a fluorescent probe can detect hydroxyl radicals. This is, kinetically, nonsense.

Look at the chemistry first.

Good luck!

Thank you for your response! I will review the article. From what I’ve read, ROS quantification can be quite complex and sometimes inconsistent. Before applying ROS measurements to my specific experimental conditions, I believe it’s essential to first assess the DCFDA assay’s performance with different positive controls.