Detection of ROS by DCFDA - (Jan/10/2012 )

I am currently doing experiments about ROS and cell signaling in two cancer cell lines.
Although application of NAC, a well known antioxidant significantly counteract the effect of my chemical, i always suffer one problem that the chemical shows no obvious influence on ROS level when using DCFDA or DHE to detect. It is certain that DCFDA works since the basic level of ROS was detectable. Is it possible that my chemical induces extraordinary ROS than the normal one?

The detail of experiment procedure is as follows:

Cells cultured in RPMI supplemented with 10% FBS and 1% P/S were treated with my chemical for 24 hours. Cells were washed with PBS, trpsinized, and neutralized with FBS. After washing with PBS again, cells were incubated in PBS containing 10 uM DCFDA for 30 min. After washing again, cells were analyzed using flow cytometry.

I had also tried to stain cells without trypsinization; however, the result remain similar with no significant difference.

Any suggestion?

Thank You

If am correct, Usually you have to incubate 10 µM DCFH-DA for 30 min at 37°C prior to the experiment, not after the experiment.