I was trying to detect H2O2 induced intracellular Reactive Oxygen Species (iROS) generation with DCF-DA fluorescent dye (2′,7′-Dichlorofluorescin diacetate, Sigma Aldrich, St. Louis, MO) in HK-2 cells. For my surprise, I was observing higher fluorescence values (using a plate reader, Ex 485nm cut off 530nm, Em 538nm) for the control groups verses H2O2 treated groups. Does anyone have an idea, what’s going on?
Thanks,
Susara
Protocol briefly:
Seeding 10 000 cells/well in a black 96 well plate, 24h incubation, H2O2 treatment 0.1, 0.2, 0.3, 0.6 and 1mM for 1, 2, 3 and 4 h. Cells were washed twice with HBSS and 10uM of DCF-DA dye in HBSS was added and incubated for 30min.
The dye was excited at 485nm and the emission was recorded at 538nm.
Hello, Susara.
That does not surprise me. Your cells are too many. The cells with limited amount of medium turned yellow, I guess. Therefore, high oxidative condition was created in doubled cell number after 24h culture in the absence of H2O2 (control). In contrast, inhibitory effects on cell division was created by added H2O2 with much less cell number. Just compare the pH of the wells after 24h treatment by putting on a A4 sheet immediately after taking the plate out of incubator.
The other point to improve the readout is that reducing the treatment groups to avoid recapturing free DCF-DA remained in the Hank's after washings to the cells again during long procedure. Also, reduce 5 to 8uM DCF-DA to give proper baseline and washing without removing cells (it would be hard).
Good luck.
Hi Dr. Lee,
Thanks a lot for the guidance. Now I see what could have happened. I will try again.
Susara
Hi Professor Lee,
I repeated the assay with several cell densities (2000, 3000, 4000 and 5000 cells/well in 96 well plate) and used 5uM of DCF-DA this time. But the fluorescence was still the same. Control gets the highest while H2O2 produces a lower.
Doing the same in a 6 well plate (cell density adjusted) revealed that the washing with HBSS removes HK-2 cells. Therefore I tripsinized the cells in each group and normalized the fluorescence values based on remaining cell number. Now it seems the vales are pretty reasonable.
Thanks.
Hi, Susara.
It seems to work, well done! From the my work, again I looked for the concentration of DCF-DA in DMSO I used. I used 300nM DCF-DA for fluorometry and 150nM for FACS for 15~20 min incubation, respectively. I realized the concentration you used and I adjusted in above answer, assuming that safer level than you are using is too high leading to cell toxicity due to both DMSO and DCF-DA
There is an issue on the DMSO toxicity and stock solution of DCF-DA. We usually make our stock 10~20mM in DMSO to minimize the final DMSO contents. In fact, in the similar topic discussions I wrote 0.5mM DCF-DA. https://www.researchgate.net/post/What_is_the_best_protocol_to_detect_ROS_in_cell_culture_using_CM-H2DCFDA
https://www.researchgate.net/post/Percentage_of_DMSO
That was wrong!!! I should have written 0.5uM DCF-DA. We decreased up tp 0.3uM (300nM) in the end.
So, the conc. you are using is far too high. Of course the cell number should be as discussed above. If you take these two issues, you will get a perfect dose-dependancy of DCF-DA fluorescence.
Best wishes.
Hi Professor Lee,
Ohh.. That is very interesting. For sure, I will give a try.
Thanks in advance.
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