Most ROS have very short half-lives. DCF is not particularly stable chemically or photochemically. It also lacks selectivity. If ROS interconverts during your experiment period, you will have concerns for variability and how to account for the dynamics or lack of it. 

For a review of DCF's problem, see: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730091/

The editorial board of the SFRBM (http://www.sfrbm.org/) recommends against using DCF for studying ROS. 

perhaps measuring H2O2 level would be more viable an option. H2O2 has the longest half-life among major ROS.

https://www.thermofisher.com/hk/en/home/brands/molecular-probes/key-molecular-probes-products/amplex-red-enzyme-assays.html

As stated by other ROS are shortlived and this is this short life (high reactivity) that explains their deleterious nature. Even H2O2 is short lived if catalase is present.

I find a little bit surprising to imagine that a "somehow measurable pool of ROS" in the homogenate would represent the oxidative stress status in the living matter rather than revealing possible interactions between the homogenate components and the oxygen of surrounding atmosphere. And this without taking into account the questionable nature of "ROS probes".

I acknowledge this is not "optimistic"! Then what I would suggest is to consider possible damages or molecules derived from ROS attack that would integrate the ROS attack in vivo. Possible parameters are:

- GSH/GSSG ratio

- Aconitase/fumarase ratio

- Oxyblot of proteins.

- modification of DNA bases, breaks...

- Oxidized lipids.

Then the history of the homogenate should be taken into account, for example in our experience the Aconitase/fumarase ratio is affected by freezing/thawing.

I hope it helps somehow.

FB

this manuscript will help you

 http://link.springer.com/article/10.1007/s12011-012-9586-3

Dear all,

Thank you very much for your answers. Indeed it seems that DCF has a lot of drawbacks and is not recommended anymore..

The problem with H2O2 is that probes like AmplexRed are not cell permeable, and the homogenization I use is only "gentle" because I assess mitochondrial functionning in parallel. One idea might be to use another homogenization technique (more efficient at disrupting cell integrity) or to permeabilize the sample with digitonin for the H2O2 assay. What do you thinks?

It seems that some probe for intra-cellular H2O2 now exists, but I find few information about such probes for the moment.

Thank you very much Frédéric for your input, even if I agree that tis is not very optimistic I already have measurement of damage and GSH/GSSG ratio planned, along with antioxidant measurements, but I wanted to have an idea of the whole balance: ROS / antioxidant --> damage.

All the best,

Antoine

Is it possible to detect ROS using DCFH-DA in non-homoggenized kidney fresh tissue? I wonder the condition that we easily dected the fluorescent of DCF.

Sang Heon Song Yes it's possible. Normally you incubate your organ with a combination of Medium (MEM+FBS+ DPBS or Hanks solution) + DCF + Hoescht for 30 minutes at 37°C, then you embed it with Tissue Tek and get the sections you want with the cryostat's aid, after that you can analyse your tissue samples either using a confocal or fluorescent microscopy. Normally you have to do everything on the same day do to the ROS lifespan.

I use DCFDA in liver tissue as described here:

Article Dexamethasone counteracts hepatic inflammation and oxidative...

Hello Dr Stier

DCFH-DA assay is the method of choice for the estimation of hydroxyl, peroxyl radicals and H2O2 from tissue homogenates by spectrofluorimetry. If you are interested in the measurement of superoxide anion along with the above, you can try DHE staining. You can also visualize the localization of ROS by fluorescence/confocal microscopy from tissue sections as well. I have used both staining methods on isolated mitochondria from various tissues and brain tissue homogenates. However, there is an issue of background fluorescence of DCF which can be eliminated by repeated washing and avoiding exposure to light during the experiment. Hope it helps.

Ilika

The mechanism of DCF oxidation is not understood in vivo. Its likely catalyzed by peroxidases though, and so reflects their activity in addition to the concentration of peroxide. I don't know of any small molecule ros probes where the interpretation is clear.

Dear Ilika Ghosh ,

I'm trying to find a way to measure ROS on human brain homogenates. Is there some literature of yours I could refer to?

Thank you for your help!

Hello Zeba Firdaus

As far as I know, that protocol can be used in different tissues including the brain, what may change is the time of incubation (since some tissues seem to be "thicker" or "harder" than others for the reactants to enter)