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!!