Flow Cytometric Detection of Reactive Oxygen Species   

Edited by
Lin Fang
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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.

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hi, I am trying some modifications of the above-mentioned protocol but still, I cant get a valid experimental setup. the problem is that both treated and control (no treated samples) give a fluorescence signal. another observation is that the fluorescence is visible even in the cell suspension so in this case, once I pellet down the E.coli cells and resuspend them in PBS (my working solution for flow cytometry) I have very weak or no signal. Could you please help me or test me if you have any recommendations?

thank you in advance
4/24/2018 3:29:49 AM Reply
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