I'm interested in looking at ROS levels in some of my cell lines, both intracellular, and levels in the tissue culture media. I am especially interested in NOx, but a technique detecting many different types of ROS would be ideal. Ideally looking for fairly cheap kits or the core chemicals - can involve flow cytometry, plate readers and fluorescent microscopy if necessary, but the more basic the better. Just wanting to have a 'quick look' currently as a possible side project to one of my more core research projects.
Agree with Benjamin Frey. 2',7'-Dichlorofluorescin Diacetate from Sigma is cheap and easy to use for measuring total ROS in live cells by Flow or Fluorescent microscopy. I typically use 2.5 - 5 uM and load the cells for 20-30 min at 37C before washing and analyzing by flow or mic.
If you want dyes that would specifically measure mitochondrial ROS, then you have to use MitoSox from Invitrogen, which works very nicely.
Dear Victoria,
Have a look at this paper ( Isoluminol-enhanced chemiluminescence: a sensitive method to study the release of superoxide anion from human neutrophils.
Lundqvist H, Dahlgren C).
there are some kits for flowcytometry too...I think from BD or Roche...
I believe the cheapest and quickest way to detect ROS in cell culture (if you're not interested in specifically teasing out what ROS species are being produced) would be to use dihydro-rhodamine in the media. I'm not sure if it reacts well with superoxide however, but it does react with peroxynitrite and other oxidative radicals. In general it gives a good sense of overall oxidation.
You can prepare it in dimethylformamide (or DMSO may work as well). Use 50uM final concentration in culture. When done with experiments, measure cell supernatants at ex: 500nm/em:570nm.
There is also a xylene orange kit for peroxides: http://www.piercenet.com/browse.cfm?fldID=02020301
One cool way to measure ROS is with redox sensitive GFP. Check out this article:
http://www.cell.com/cell-metabolism/abstract/S1550-4131%2811%2900427-X
Agree with Benjamin Frey. 2',7'-Dichlorofluorescin Diacetate from Sigma is cheap and easy to use for measuring total ROS in live cells by Flow or Fluorescent microscopy. I typically use 2.5 - 5 uM and load the cells for 20-30 min at 37C before washing and analyzing by flow or mic.
If you want dyes that would specifically measure mitochondrial ROS, then you have to use MitoSox from Invitrogen, which works very nicely.
Use DPPH radical scavenging assay [Stable N radical], epinephrine auto-oxidation inhibition assay, hydroxyl radical scavenging assay by using Fenton's reaction etc.
all these assays requires chemical that are easily available and cheap.
Thank you very much for all of your replies. Much appreciated. I have ordered some DCFH-DA and will do some preliminary experiments next week.
Hello, I was thinking to perform ROS measurement as well but not sure which probe to choose. Does the DCFH-DA they recommend work well? Does it measure intracellular or extracellular ROS? Could you please share the protocol?
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