H2DCFDA for MDCK cells - (Oct/27/2013 )

What is the best protocol to detect ROS in MDCK cells using the confocal microscope?! I tried to do time lapse for 10 minutes and noticed what is look like cells are dying or signal is disappearing within minutes!! Is it possible to do time lapse with H2DCFDA for MDCK cells?!

Please see attached pictures for ROS intensity signal for untreated MDCK cells over 10 minutes (notice the decrese in grey intensity; z0, z1 to z3), cells are seeded for 48 hours, after that H2DCFDA is added (I tried different concentrations; 10-1 uM and the pictures are for 1 uM concnetration) for 30 min and then I wash my cells 3x with PBS 1x. Any suggestion!!

I've had "experience" with several ROS fluorescent dyes, both in confocal and in FLIPR assays.  They have never produced the expected data. In confocal studies, the dye bleaches quickly as you seemed to have observed.  This makes it very difficult to detect changes in ROS for biological or drug agents that are known to generate them. Adding ROS donors as positive controls will work, but when you're looking for small changes caused by agonists, you won't see anything.  I wish you luck with this technique, but I'm not sure that you will be successful.

Tom M on Wed Oct 30 17:32:07 2013 said:

I've had "experience" with several ROS fluorescent dyes, both in confocal and in FLIPR assays.  They have never produced the expected data. In confocal studies, the dye bleaches quickly as you seemed to have observed.  This makes it very difficult to detect changes in ROS for biological or drug agents that are known to generate them. Adding ROS donors as positive controls will work, but when you're looking for small changes caused by agonists, you won't see anything.  I wish you luck with this technique, but I'm not sure that you will be successful.

Thanks for your reply, in addition to what you mentioned, it is difficult to obtain consistent results, 1uM of the dye used to give me a strong signal but today I got a really very weak one, I will try to use the plate reader to measure fluorescene intensity instead of imaging with the Confocal.