Flow Cytometric Detection of Reactive Oxygen Species   

Edited by
Lin Fang
Download PDF How to cite Favorites 1 Q&A Share your feedback Cited by

In this protocol

Ask a question to the author

Original research article

A brief version of this protocol appeared in:
Cancer Research
Sep 2012


Reactive oxygen species (ROS) are molecules containing hydroxyl radicals or peroxides with unpaired electrons. In healthy aerobic cells, ROS are produced naturally as a byproduct of oxidative phosphorylation, oxidoreductase enzymes, or metal catalyzed oxidation at a controlled rate. However, ROS can be induced under some stress conditions especially exposure to environmental oxidants and certain drugs that leads to oxidative stress. Exceed ROS can cause damages in the building blocks of cells including DNA, proteins, and lipids, and eventually results in cell death. Cell-permeant 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA) is a widely used ROS indicator. The reduced non-fluorescent fluorescein H2DCFDA can be oxidized and converted into fluorescent 2’, 7’-dichlorofluorescein (DCF) by intracellular ROS. In this protocol, we applied H2DCFDA to label the intracellular ROS and detected the DCF intensity by flow cytometry.

Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Chang, H., Huang, H., Huang, T., Yang, P., Wang, Y. and Juan, H. (2013). Flow Cytometric Detection of Reactive Oxygen Species. Bio-protocol 3(8): e431. DOI: 10.21769/BioProtoc.431.

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.

You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Rodrigo Hoyos
Boston Children's Hospital
Hi, I'm trying to set up your protocol and I wanted to confirm if NAC/H2O2 were present in the cell culture media while the cells were getting to a confluency of 70-90% (for positive and negative controls respectively) and after cells were treated for an adittional hour. (Steps 2, 3 and 4 of your protocol). Thanks for your help.
12/3/2015 8:29:54 AM Reply
Hsin-Yi Chang
Kyoto University

Hi, I'm not sure what your question is. According to the protocol, cells were treated with NAM or H2O2 after reaching 79-90% confluency. At the end of treatment, cells were trypsinized and incubated with dye containing DPBS.

12/4/2015 3:32:51 AM

Rodrigo Hoyos
Boston Children's Hospital

Thanks a lot for your answer, that's what i figured but I got confused since in step two it says "Cells were treated with NAC or H2O2 until 70-90% confluency reached" instead of after reaching 70-90% confluency.

12/4/2015 5:40:30 AM