Does anyone know which ROS (super-oxide, hydrogen peroxide, hydroxyl radial, mitochondrial or cytoplasmic) is targeted by the CellROX Deep red reagents? In the specifications of the product, it is just said that ROS can be measured without any details.
Hi, Nicolas, the cellRox Deep red is an intracellular dye and fluoresence dependent from the oxidation status. Despite they did not mention which type of the ROS you can detect, I suppose that this is an hydrogen peroxide, rather than superoxide. Good luck!
Thanks! That is what I also deduced. I guess I should try the MitoSox instead to measure the mitochondrial superoxide...
Yes, exactly! Mito8Ox will be for mitichoindria. You can also try DHE if you want to be more specific for intracellular superoxide accumulations. Please, also remember that washing step with new medium (like they mention in many protocols) may serve as stress-inducer itself. Try to minimize washing or even avoid this step. Good luck!
Virtually any of the dyes you mentioned, DHE, MitoSox, DHR, DCF, and all their variants will measure ROS in live cells with fluorescent microscopy. None of them are truly specific for single ROS specie in live cells. In vitro/test tube, yes.
In order to measure a specific specie, you also have to add membrane-permeable reagents to remove that specie. For example, if you want to measure cytosolic H2O2, you can overexpress catalase or treat with PEG-catalase. If treatment group shows increased fluorescence, but treatment+catalase shows less increased fluorescence, the difference between the two is caused by H2O2.
For superoxide, see works by Kalyanaraman's group at MCW: http://www.ncbi.nlm.nih.gov/pubmed/20116425. The superoxide-specific DHE product needs to be separated with HPLC then detected. You might be able to overexpress both SOD and Catalase to zero out the signal and prove it's superoxide specific, but that's a lot of work for 1 claim.
I recommend not spending the effort to identify particular ROS specie, unless if it would really add significantly to your paper/grant.
I got the attachment from an Invitrogen product rep. from their assay of the fluorescence increase from different ROS/RNS sources.
According to it, CellROX fluorescence is in response to (in order of intensity) 1. hydroxyl radical, 2. superoxide, 3. TBHP
We did confocal imaging of CellROX in diff. cell types w/in hippocampal slices (Grinberg et al., 2013). It turned out looking pretty good; we were even able to go back an re-image over a month later, so I guess it is pretty photostable (in fixed, Antifade-coverslipped tissue).
Very good paper Yelena. Do you think CellROX will work on fixed spinal cord tissue (naive and contused)?
Samirkumar, in short, no.
I have seen one publication that does something like this, with the idea being that the ROS-producing enzymes are still there, and if in one paradigm (e.g. contused) there are more active enzymes then you can use CellROX to capture their production of ROS in a sort of in vitro assay. I just don't think this is a good idea; you're altering enzyme kinetics if the tissue is fixed. This seems needlessly complicated. There are better ways of assessing ROS-producing enzyme activity/activation, or simply staining for oxidative stress via something like MDA.
Nicolas, the signal obtained with a probe like CellROX in cell culture will depend on the response to the different ROS (measured in solution), but we must also take in account the different sites and rates of generation of each species, as well as their reactivity and diffusibility in the cell environment. Superoxide has a very low half-life in cells (some micro-s), while H2O2 is more stable and may diffuse through membranes, but gives a lower signal with this probe.
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