Dear Reinold,

Below is my protocol for DHE staining fly tissue. While I understand you're using cells this might give you an idea of a good protocol:

1) With tissue in growth media (we do fly dissections in M3 for example) add 10uM DHE, nutate at room temp for 7min and protect from light

2) Wash 3x5mins in growth media

3) Fix, 8% PFA for 6min

4) wash 1x PBS and mount/image.

DHE is really picky in that every step needs to be at a consistent time for results to be comparable. For this reason it's best to also test the control at the same time if your experiment allows. In my experience, the fixation step is not super required but does give cells better boundaries when imaging. DHE stocks also tend to expire rather quickly so be careful of the stock solution your using. Alternatively you could try CellRox from LifeTech, the green version has the added ability to be multiplexed with stains and the signal is much clearer than DHE.

Hope this helps,

--Ryan

Thanks Ryan, much appreciated! I'll use the concentration 10uM and see if it works. I'll be running the cells through a flow cytometer though so no fixing.  

Please check my paper, Morshed SA et al., J Autoimmunity 2013. Once adherent cells are confluent, wash them with HBSS 2-3 times carefully so that they don't get detached. Dilute the dye to appropriate conc. in HBSS, add, incubate at 37 for 30 min. Wash again with HBSS 2-3 times, you can now read them in fluorescent reader and also watch them under microscope. Thanks.

I have work for doctorate thesis with DHE for ROS evaluation. This dye is more sensible to the light, so attention when prepare the stock solution, to must have a dark environment, not is easy detectable because duration in time is between 5 to max 30 min because decade. For the wash not plus of 15 min as describe in your protocol can use Phosphate Buffer Saline (PBS) at ph 7.4 for cells.  DMSO is toxic substance for cells so 30 min not is good for vitality of cells and of course to obtained good answer in ROS formation so you can try use for dissolved the dye PBS or HBSS since DMSO. Hope  This helps,

Annarita

Thanks Syed and Annarita

Syed, do you use a concentration of 50Um DHE as stated in the paper or less?

Hello, what's the cell type you are using? 

If macrophages, either primary cells (primed for a week) or cell lines like RAW264.7, you can try LPS (200-500 ng/mL) plus interferon gamma (20-50 ng/mL), for 14 to 18 h. This time zone will give you heightened response for ROS production. 

LPS at 30 min to 1 h as a treatment does not induce clear change. 

See these ref attached. 

Article Molecular Imaging of Peroxynitrite with HKGreen-4 in Live Ce...

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

Thank you Nai-Kei! The cells are retinal cells. Is the interferon gamma necessary (I haven't been adding it to my treatments, just plain LPS)? I had one treatment of 24 hours but still did not detect any increase in ROS production. I might try treatment with the interferon and see if that makes any difference.

Basically LPS + IFN-gamma is a classical combo with synergistic readout. It's good for macrophages, but may not be best for other cells. 

Elsewhere, people put in TNF-alpha as a third stimulant (in range of 20-50 ng/mL). Stand-alone or in combo.  

Also, nutrient supplements like pyruvate is going to suppress the respond (I have done tests to find out) of ROS production, potently. Pyruvate is a well known endogenous scavenger for H2O2.  I usually take out pyruvate supplement from the culture. 

In my experience, 24 h is not necessary a good time point for readout. Instead, 12 to 18 h is better.  

Because different cell types respond differently in kinetics and intensity, you may need to find out. 

If you have cytotoxicity results, they will suggest how long the cells stand before losing viability. Just before cell death, there's usually also a window for observing increased ROS. 

Yes, but it depends on cell types so you need to optimize the concentration. You can start with 10microM and then increase by two-fold when you see good expression and less background.

Hi I picked up a very similar project, never having used DHE or LPS before, using non professional macrophages, J774A.1.  Iv tried optimizing different concentrations for live imaging and end up using 15uM of DHE for 20 minutes after LPS (1ug/mL) incubation of 1 to 2 hours (washing in DPBS after both).  This showed to have a significant florescence increased compared to no LPS.  Therefore, I've been sticking with it.  Message me if you'd like more details.  

A prior commenter here suggested fixation, but I would not suggest this. DHE does not contain primary amines that could be crosslinked by aldehydes. Also, even if it could be retained, it will not preserve the same signal after fixation that it had before fixation. In fact, you would more likely be reading the ROS of your buffer at that point.