I notice that your sample images contain fixed cells. Under this condition, there's no way for fixed cells to produce ROS (notably superoxide, then H2O2) in logically predictable ways.

It's more likely the fluorescence increase is due to auto-oxidation of the probe, in the presence of laser photon. In other words, photo-activated auto-oxidation. 

This makes the probe not to be called robust enough for imaging. 

****

Our lab has just published a JACS paper on superoxide detection. 

You may consult your supervisor, whether it's desirable to write and request the probe from our lab. We distributed a requested aliquot to a US lab, just one week after the paper was published. 

However, the decisions of whether to grant the request rest on my boss' discretion.... :)

Article Fluorescent Probe HKSOX-1 for Imaging and Detection of Endog...

If you have same strong signal in control cells maybe your CellROX Green reagent is already oxidized (not good store conditions, not protected from light, etc.).

did you follow all recomendations https://tools.lifetechnologies.com/content/sfs/manuals/mp10422.pdf

I agree with Dr. Petrenyov. 

One way to check if probe dose is optimal and if photo-activation (turn-on by strong laser alone) happens, is to do a time-series experiment in confocal imaging with a few different doses of probe in non-drug treated cells (unstimulated), and then observe whether, over time, the fluorescence goes up very fast (e.g. within 2 min). 

You can vary the laser intensity, so as to see if  some photo-activation can be minimized.

An overdose of probes (meaning a surplus of it) also contributes to higher likelihood of photo-activation.

Green emission region is prone to autofluorescence interference.The excitation wavelength tends to be short, to give more energetic laser bombardment.

There is a Deep Red version of CellRox, which may be more robust.

**** 

 In addition, if you used H2O2 as the treatment, there should be a wash step before your probe incubation. It is not clear if CellRox reacts or does not react with H2O2, which may confound your readout. 

Best

Dr.Petrenyov and Dr.Nai-Kei Wong, thank you to the appropriate advice.

Cell ROX Green is just purchased, it has stored at -30 ℃. So, we think that the reagent's condition is not so bad.

Even in the fixed cells, green signals become to be strong more and more with about only 1 minute.

Instability of this fluorescence is strange for me.

Hello,

maybe you can try roGFP2 ?

I add a picture that CellROX Green is stained.

I notice that your sample images contain fixed cells. Under this condition, there's no way for fixed cells to produce ROS (notably superoxide, then H2O2) in logically predictable ways.

It's more likely the fluorescence increase is due to auto-oxidation of the probe, in the presence of laser photon. In other words, photo-activated auto-oxidation. 

This makes the probe not to be called robust enough for imaging. 

****

Our lab has just published a JACS paper on superoxide detection. 

You may consult your supervisor, whether it's desirable to write and request the probe from our lab. We distributed a requested aliquot to a US lab, just one week after the paper was published. 

However, the decisions of whether to grant the request rest on my boss' discretion.... :)

Article Fluorescent Probe HKSOX-1 for Imaging and Detection of Endog...

Try working in black walled clear bottom plates as well; clear plates themselves can result in large auto fluorescence. An aside, we recently tried Amplex red which detects H2O2 easily if you are interested in that specific ROS.

Hi Laura

Can you explain in detail what is the role of clear bottom plates to cause auto fluorescence and does this applicable to other kind of ROS detection assays that uses fluorescent dyes. If so How to make the black walled clear bottom ? or is it available like that. Thanks in advance.

Corning sells such black-walled plates: Costar 3603 (black). 

http://catalog2.corning.com/Lifesciences/en-US/Shopping/ProductDetails.aspx?productid=3603%28Lifesciences%29&categoryname=

I have found the same trend to be true.

We have been time lapse imaging fibroblasts using CellROXGreen, exposing to light 250 ms every 3 minutes over a 45 minute time period, and if I extend the light exposure to focus or adjust for too long the cells are much brighter. I assume this is the autooxidation mentioned above, but wanted you to know you're not alone in this!

We are still troubleshooting CellROX, trying to find the best assay for detection. It has not proven to be simple.

We are using CellROX deep red reagent on primary porcine fibroblasts. After treating the cells with Cell ROX reagent we scan the cells each 10 minutes for 2 hours using fluorescent spectrophotometer. Afterwards we treat cells with Hoechst. The signal is then normalised to Hoechst staining and the Change in Cell ROX intensity / Hoechst is calculated.

We recently try Cell Rox Orange for OS detection on brain ex vivo explants. Although fluorescence microscopy is one of the compatible techniques they mentioned, for us it didn´t work. The immense increase on flourescence seconds after the sample is placed on microscope suggests that the probe is not robust enough for this determination, similar results have been describe even on fixed cells. https://www.researchgate.net/post/Please_teach_us_how_to_detect_oxidative_stress_using_CellROX_reagent?_ec=topicPostOverviewFollowedQuestions&_sg=IU3juzgGKtQKIZWTDfXLInDz2MwI0cl8o9dkbDCHY8S3S4DQ88gHlHroataKyBJNu6FHMnejTdZIgCCN.SWwurpS8uZWsbfAYId3ENzRXx3CNPn4WzSPz3XOmOTja57psspl6qBLqptjdjTKjCSdrweUmMkucx6sQ3bdCSLs