Measuring intracellular ROS using DCFDA is creating lots of misleading data, I would really like to switch to other protocols available to measure ROS. But my point is where should I really look for? Intracellular or Extracellular ROS?
What if I use Amplex Red?
Can I justify my compound's antioxidative property with this data?
As I've said in a few questions, do not use DCF at all. It is useless. Amplex red will measure extracellular H2O2. The problem with this is only up to around 15% of H2O2 produced intracellularly gets outside the cell, so if your cells are producing only a modest amount of ROS you may not see it with this assay. Try dihydroethidium, for intracellular superoxide. I gathered earlier that you don't have access to EPR (this is the best way to measure ROS).
Dear David,
Thank you . We have been planning to go for Amplex Red, but yes of course considering the point about how much we could get from the assay. Certainly DHE would be a better choice but technically its not feasible right now. And we are interested in real time response from cells, dont really want to perform with supernatant. Couldn't really justify which would be the best way to follow.
Looking forward to have some response here.
Thank you again.
