Thanks Alaaeldin, I agree that could be the reason. However, this finding would be in strong contrast with the repeated and published observation that the antioxidant cocktail I used prevent the loss of sperm motility, the increase of lipid peroxidation (assessed both in situ and biochemically) and the increase of DNA fragmentation seen in parallel sperm incubated in the same culture medium without antioxidants (Talevi et al 2013, http://www.ncbi.nlm.nih.gov/pubmed/23958080)

Hi Roberto, do you use flow cytometry?, we have observed a fluorescence increase in patients taking antioxidants. It seems that antioxidants might be interfering fluorescent reading. Even in unlabeled cells,..

Hi Rafael, I use epifluorescence or confocal microscopy. I wonder whether oxidants and antioxidants can interphere with the activity/fluorescence of cell rox green. Do you have an increase of fluorescence after antioxidant therapy even in the absence of cell rox?

We use cytometry, and sometimes we see autofluorescence without adding any fluorochrome. The conclusion: it could be due to antioxidant treatment because many of our patients is done. It would be interesting to check.

Small molecule antioxidants can potentially generate ROS. For example flavenoids, and quinones generate semi-radical intermediates which an potential generate proximal ROS like superoxide. Rates of ROS production by flavenoids and quinones can be quite high reaching up to 10^8 M-1 s-1. Small molecule sulfur containing antioxidants like GSH can also produce ROS through the formation of thiyl radical intermediates. In fact genesis of a GSH radical can lead to the formation of GSSG radical which can then lead to the formation of superoxide and hydrogen peroxide. The rate is astoundingly fast (10^8 M-1s-1)

GSradical + GSSG -> GSSGradical 8 x 10^8 M-1 s-1

GSSGradical + O2 -> GSSG + superoxide 2 x 10^8 M-1s-1

The superoxide is then dismutated by SOD to produce H2O2. This is called the electron sink hypothesis.

Of course for an antioxidant to serve as a prooxidant as described above, the concentration must also be high as well.

Thanks Ryan for your answer. However, as I previously told, the increase of ROS detected through cell rox green in human spermatozoa treated with zinc and coenzymeQ10 would be in strong contrast with the associated effects on prevention of motility and viability loss, lipid peroxidation and DNA fragmentation. As in a previous discussion it has been reported that invitrogen advice to not use H2O2 as positive control as it quenches the cell rox fluorescence, I wonder whether a stronlgy reducing environment in the reaction mix may preserve the activity of the dye and produce an artifactual higher fluorescence in the cell. Several researcher had a lot of trouble and unexpected findings using this probe that were sometimes solved using fluoresceine diacetate for ROS detection. I selected cell rox green as is it resistant to fixation and permeabilization thus allowing the correlation of ROS intracellular levels with other markers on the same cell.

Hi Roberto, That is interesting considering that Invitrogen reports that Cell Rox Green is weakly fluorescent under reducing conditions and exhibits increased fluorescence upon oxidation. Based on your description it sounds that the dye itself needs to be stored in a highly reducing environment and all assays performed in the dark with reductants present (like DTT, TCEP, and mannitol). It is puzzling that H2O2 would quench fluorescence since it is an oxidizing agent. One has to question if Cell Rox Green is suitable for detecting oxidation.

Hi Ryan, the paradox of fluorescence quenching by H2O2 might be that with H2O2 in the external environment the dye fluoresces and then there is less dye ready to fluoresce within the cell. This is just a speculation and really it could be better to adopt another dye

Hi,

- I think the comments suggesting increased ROS-generation by Zinc/coQ10 are not necessarily in conflict with your previous work showing decreased lipid peroxidation and DNA damage. Since CellROX detects ROS and not oxidative damage, the CellROX molecule may be seeing intracellular compartments that generate ROS whose half-life is too short to do any damage due to compensatory antioxidant mechanisms (e.g., the tightly controlled ROS elimination in mitochondria). Just a guess.

- I worked w/ CellROX deep red, and its protocol instructed using formalin as the fixative. We observed false-negatives with 4% PFA; I realize this may be the opposite of what you are observing, but in our hands the fixative had a significant effect on Cellrox fluorescence (2013 J.Neurochem.). So, if positive/negative controls aren't working, this may be the culprit.

- What was the protocol that allowed use of H2O2 as a positive control? Did you apply H2O2 and Cellrox simultaneously or sequentially and at what dose?

- Cellrox definitely has its drawbacks: conflicting H2O2 responses; proprietary molecular structure that precludes a decent educated guess as to where it's going within a cell (i.e., does it have a lipophilic component?), and I'm sure others. But Cellrox does allow for tissue fixation and its fluorescence is quite stable, so it allows for great fluorescence IHC unlike most (any other?) ROS probes. I was interested in cell type-specific ROS production in a heterogeneous tissue, and in that scenario Cellrox worked very well. I think coupled to other forms of ROS or OS detection and some healthy skepticism Cellrox can provide meaningful data. Here it seems that another ROS detector can be easily substituted.

Hi Yelena,

thanks for your useful comments. I red your paper 2013 and the influence of fixative in the detection of a basal and oxidated condition of the tissue. We used 4% PFA to better preserve the reactivity for labeling with other probes. However, I'm going to test just phosphate buffered formalin and do positive controls that just induce ROS within the cell. Your guess on the higher intensity of cell rox in cells treated with zinc/CoQ10 that are healthy compared to untreated cells is surely interesting but possibly hard to demonstrate. A problem when working with sperm suspensions is that you never completely eliminate H2O2 or Zinc/CoQ10 before cell rox labeling. Reducing their concentrations involve multiple centrifugations that are a source of damage and oxidative stress for sperm. As concern the localization of the signal it is not necessarily located where the ROS are produced as cell rox green, differently from deep red, once oxidizied binds DNA and localize to the nucleus and mitochondria. I think, as you suggested, it is also worth to test other dyes as CM-H2DCFDA to confirm Cell rox data.