Two hour incubation is awfully long in my opinion. Here's my protocol for DCF-DA analysis of adherent cells:

- Wash cells with 1x HBSS

-Incubate with 20uM DCF-DA (HBSS) for 15-20mins at 37 degrees C.

-Trypsinize cells, inactivate trypsin and suspend cells using HBSS containing 5% FBS. Then centrifuge.

-Re-suspend pellet in HBSS to desired concentration of cells and analyze. (Obviously keep the DCF-DA treated cells in the dark at all times)

Ex/Em 492-495/517-527

Hi Lilli, something must be wrong with diluted DCFH. What do you use as diluent? 1uM concentration usually, is not toxic. Other thing is to incubate your cells for 2 hours without medium, then is normal that cell die. Check your diluent, best regards, Alberto

 Hi Lilli,

I think you could try a shorter incubation period. Usually half an hour is enough. Best! 

To much organic solvent may be presented in final solution. What is your stock solution concentration? It should be 1000 fold (1 mM) or higher, so in final solution solvent (e.g. DMSO) would be presented in minimal amount.

Hello!! I think you should check the stock solution of dcf and the final concentration of dmso if you use it as a solvent. I worked with spermatozoa which are extremely sensitive to any handling and i used dcf in concentration of 20uM. Do you exclude dead with pi? If the pi is not fresh then there could be a misinterpretetion. Did you stain the cells with trypan blue for example to see that they are dead? Maybe you should also check the settings of fc and medium composition and sterility. Here,you can find my protocol https://www.researchgate.net/publication/280255455_Antioxidant_effect_of_crocin_on_bovine_sperm_quality_and_in_vitro_fertilization

Article Antioxidant effect of crocin on bovine sperm quality and in ...

 2 hours is a lot of time. I used 20 mins of incubation time. The kit I used for ROS can be found in this link http://www.enzolifesciences.com/ENZ-51011/ros-id-total-ros-detection-kit/

Thank you all!

I found the problem: As I said I used RPMI serumfree medium. I found out that not the DCFDA was the problem but the medium. Now I used RPMI with reduced 5 % serum containing medium and my cells survive :)

THANK YOU A LOT !

Hi Lilli,

For ROS measurements, you can not use Serum containing media. Factors in the serum will degrade most kind of ROS.  I would follow the above suggestions of 20 mins incubation and Mathew  and others gave an excellent protocols for you to try.

Thank you but I have excellent difference between treated (1mM h202) and untreated cells when I culture them in serum reduced media :) In PBS my cells are dying..

Try using serum free media. 5% of serum will quench your H2O2. Reviewers won't like any serum in the media. I hope this helps

 Hello Lily. I suggest you to prepare a standard solution of your substance and to see its toxicity below 1 micro M. Then to use the quantities that have low toxicity and to follow the normal protocol. Βut you having in mind that many free radical scavengers have their own toxicity, whereby the toxicity curve is necessary. Good luck and if you have trouble talking again.

Hello Lily, Hope you have good time. I had the same problem some time before using DCF for ROS detection. But I think you try as 

1. When you need to perform your experiment for DCF, just remove the culture medium and wash once slowly with serum free medium to remove the dead cells and cells debries (Dont use PBS). Incubate your desire cells in serum free medium for about 30 to 40 min in dark (with a 5 to 10 μM DCF), I normally use 5μM DCF and getting good results.

2. Some cells have low level of ROS so use some ROS inducing agents such as TNF alpha or CoCl2 etc.

Hope these experience will work for you too

3. If these experience not working for you then please change your DCF reagent and try another one to compare your result (Some time DCF get some problem either because of longer time or mixing with other reagent or not proper storage)

Good luck

Hi!!

I had a similar problem with some of my cells that were very sensitive to incubation with PBS and we moved to incubate the probe in HBSS without phenol red. Viability improved a lot!!

Good luck

In addition to great suggestions given above, the cells you use matter as well. Eg neutrophils die easily when using simple buffers such as HBSS. Loading for 2h is unnecessary. usually, 30 minutes suffice. Also, depending on the cells, try to keep DMSO concentrations below 1% or even .1% for some cells, which should be easy to achieve as stocks of DCFDA can be made up at 10 mM. I usually load the cells at a higher concentration, eg 10^7/ml.

What type of solvent/vehicle are you using to expose cells to DCFDA? I think you need serum if you are using PBS o HBBS to give support ot the cells