You can also do DCF-DA evaluation using a plate reade without detaching the cells, which would be even better for your situation, as you can test many samples in one go in a 96 well plate, with less reagents..

If you want to use only FACS, then trying to use different lower concentrations of Trypsin/EDTA to establish the lowest ROS produced. Also try to reduce the number of washings & time, further you can keep the cells on ice, immediately after detaching. Scrapping is not good for FACS. I hope you do not wash after adding DCF-DA, as it is not required.

BW.

You can also do DCF-DA evaluation using a plate reade without detaching the cells, which would be even better for your situation, as you can test many samples in one go in a 96 well plate, with less reagents..

If you want to use only FACS, then trying to use different lower concentrations of Trypsin/EDTA to establish the lowest ROS produced. Also try to reduce the number of washings & time, further you can keep the cells on ice, immediately after detaching. Scrapping is not good for FACS. I hope you do not wash after adding DCF-DA, as it is not required.

BW.

Thank you for your comments. I was also considering of using 96 well plates, but then most of the articles, I read were using flow cytometry as the end point detection method so was considering it.

Regarding the washing step I had read that washing helps reduce the background florescence so I have been washing once in PBS after incubation in DCF-DA. Just as I search more, i can find different opinions which might have arisen in different context just difficult to find which one works the best. Thank you for your time.

I am also about going into this process. I will advice you reduce time of incubation with trysinisation and watch with PBS before analysis.Also, there are reports that DCF-DA accumulate in the cytosol of cells thus it can result in cytotoxicity of the cells in question, it is important to know the concentration to be introduce to each cell types. Mostly is it advce that u use 1-10uM for 45-1hr. And lastly hope you used serum free media because serum contains endogenous esterase activity and it will de-esterified dichlorofluororescein (DCF) and give conflicting data. Wishing you the best

Thanks John, yeah I try to keep the time of trypsinization to the lowest possible just enough to dislodge the cells, and once free give them enough serum, which i then discard to suspend the cells in PBS so that the medium is essentially serum free to decrease the conflicting data. I have tried using 50uM DCF-DA for 15 mins at 37C, since the protocol calls for time variation from 5-60mins. May be decreasing the time for incubation will also decrease the chances of cytotoxicity by the agent.

Thanks again.

@Sanjaya I think 50uM seems too high a conc.- Check this article; MEASURING REACTIVE SPECIES AND OXIDATIVE DAMAGE in vivo AND IN CELL CULTURE: HOW SHOULD YOU DO IT AND WHAT DO THE RESULTS MEAN?- Berry Halliwell and Mathew Whitman- British J. of Pharmacology (2004)142, 231-255. all the best

@Zemin I think I should try reducing the concentration of dye for sure. I will try following ur advice too. As to positive control I did not get the point and have not spotted glucose being used as reference anywhere...could you pass me the paper that says so.

@John: I tried going to the link, but then I could not download the complete article, could you please pass it on to my mailing add [email protected], it would be a great help. Now that I am already at it and stuck in the middle. I would like to get over the steps as soon as possible. Will be looking forward for the paper, hope you have it.Thanks for all your help.

Regards

Sanjaya

thanks

@John can you plz send the article to me also? [email protected] Thank you.

@Sanjaya, may be you can stain some cells, divide into two tubes, analyse one directly in FACS, other after a wash. In this way you can know in your settting, which condition is better, in terms of ROS and background.

@Aaron Thanks, Yeah I normally use the EDTA solution in Ca2+/Mg2+ free PBS itself.

I again came across one problem, the ROS in my control cell is pretty high. I have been using serum free medium for differentiation of my cells and that culturing the cells in serum free medium causes more ROS production in cells through mitochondrial ROS modulator 1 (Romo1). Also it may be the effect of Trypsnization of cells, is there any such data which shows that DCF-DA loading should be carried on after certain time of trypsinization so as to decrease the ROS levels caused by trypsinization. Thank you, will be looking forward for more new ideas from you all. thanks again

@John thanks for the articles

@Vinoth yeah may be I shall give a try, on it however, by now I have evidence which shows washing is more useful, from my data and data obtained from fellow lab mates.

Recently i had the same problem (shift to right)...control higher than H2O2-treatment.

I don't know why.

Yea I had the same problem in some samples, despite trying different solutions did not work out, so I just left it for now. Also the inability to reciprocate the results in similar works made the problem higher. I guess it might be due to the difference in time following the excitation in cells. After I failed to get the good results in my lab, had to go to another lab and had to wait sometime there so the reading in the samples varied highly. May be there are other variables behind but I guessed that to be one among the others.......Gluck with your work.

Hi

I also want to use DCF-DA to detect ROS production.

But in contrast to the other people I would like to stain organotypic slices and monitor the fluorescence via epi- or confocal microscopy.

Up to now I had major problems to identify a suitable staining procedure. I tried bath staining, but I had the impression that at least superficial cells swell. This problem was partially overcome by local pressure application via a micropipette. Since I am interested in both I want to stain neurons and glial cells. But only neurons apperared to be DCF positive even upon H2O2 application... Does any one of you have experiences concerning these problems.

Another thing that bothers me is the analysis of data. I want to check the ROS production caused by a chronic treatment, thus I have to compare control slices versus treated slices and can not use the slice itself as internal control. This gives me a hard time because DCF is not ratiometric and the staining intensity can not be controlled. I thought about checking dye concentrations within each slice via complete oxidation with H2O2, but I am not sure whether this is an appropriate approach!

I would be very happy to get some input!

hello Claudia, I myself had once tried using fluoromicroscopy techniques with DCF-DA, and the problem that appeared is that, the fluorochrome itself gets excited by the laser light, and as you observe the cells, the intensity of fluorescense increases on its own, even using very short exposure periods. I have seen images publihsed using fluoromicroscopy, may be I was not good enough at it and could not have control over self excitation. If you have no problem with that go ahead, if not consider this too. Good luck. I have not worked with slice cultures, hope someone might reply you later.

Do note the amount of H2O2 required may be quite low for ROS production and see the concentration you are using.

I used DCF-DA in my work with adherent cells, check the protocol in my paper and if you have question, ask me:

Neurochem Int. 2011 Aug;59(2):297-308. Epub 2011 Jun 16.

The extrinsic and intrinsic apoptotic pathways are involved in manganese toxicity in rat astrocytoma C6 cells.

Alaimo A, Gorojod RM, Kotler ML.

Source

Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EGA Buenos Aires, Argentina. [email protected]

A few people in this discussion said that they saw lower levels of ROS in positive control. THis is very normal when using toxic levels of H2O2 (sometimes even 15 min is sufficient to cause toxicity for some cell lines) that were not optimized. I recommend using several concentrations of positive control (H2O2) and optimize the highest level that gives a good positive response. When cells start to die, they have lower levels of esterase activity, lower DCFH, the nonflurorescent substrate for ROS to act upon contributing to a perceived lower ROS levels. Lower levels of DCFH (due to lower esterase activity) causes a perceived loss of ROS.

Hi guys, I have got a question related to these topic. what is the effect of DCFH on cells without any treatment. is there any reading with microplate reader?

Yes

Does any one used the DCF-DA from invitrogen lifetec product for ROS measurement by flow? or which DCF-DA is the right one to use for flow?

I had the same problem as well, initially. But i think it depends on the concentration of the dye, incubation time and washing. Use a low concentration (10uM) and incubate for 10 min. Then wash with PBS 3-4 times with 5min interval. It should remove the binding of the dye in the controls.

My question, I am planning to use DCF-DA to measure ROS in wound of a tumor slice, does the procedure work the same as mentioned in the protocol? Or do I have to make some modifictions? have any body used it before on tissue slice is possible on tumor slice? Hope to hear from you guys.