4 replies to this topic

[aggronor]

Hi all,

I will be performing the DCF-DA assay (from Molecular Probes) for intracellular ROS generation. I have a question regarding storage. The protocol says I should make a new dilution of working solution for each use. So after reconstituting the probe (with DMSO, in my case), should I just make aliquots of the stock solution and store it at -20? Just want to make sure this is ok and that the probe activity will be consistent and won't be compromised like this. And to what degree does it need to be protected from air? The protocol recommends dry argon or nitrogen, but is this really necessary?

Thanks!

Edited by aggronor, 07 September 2011 - 10:41 AM.

[bob1]

As the compounds you are using are being used to measure reactive oxygen species, they are reasonably sensitive to the presence of oxygen.  If you want them to keep and perform consistently, you should store them under N2 or argon.  However, if you can tolerate them going off over time (even when frozen) then they will be fine at -20 without N2 or Ar.

[Seng]

Dear Aggronor,

I'm Mr. Wu, I am going to use the DCF-DA assay too for measuring the intracellular ROS generation on in vitro monoculture and co-culture later. Although, I am not able to answer your question, but I have got question intend to ask you. Hope you could help.

For the DCF-DA, what fluorescence exitation/emission wavelength you use? 488/520nm? If you my fluorescence plate reader just have 480/520nm, is the fluorescence plate reader still can use for measurement? Will it affect the real result?

Hope you could share.

Thank you.

[aggronor]

Our fluorescence plate reader just has 485/520 nm, which works fine. 480/520 should be ok.

[Seng]

Thank you so much! Can you kindly leave me your email address? Can we be a friend? I do hope if later have any question can refer to you.