Problem with quantitating ROS using CFDA homologue

Wayne Harris waharri at
Wed Jan 9 14:59:44 EST 2008

Hello fellow flowers

In our lab we have been trying to develop an assay to quantitate ROS  
using CM-H2DCFDA for cells treated with drug using a BD Canto..  
According to the data, we are getting a very high background in  
untreated cells that have been simply loaded with the dye. When we  
leave these cells alone for an hour or two this background  
dissipates. Compare this with treated cells which also show high  
signal with drug, however in this case the signal is maintained up to  
two hours later. We have also looked at these samples using a plate  
reader and found that there is signal if we mix dye and media  
together, the background on the untreated cells is much greater in  
this instance also. It should be pointed out that when the dye is  
being loaded the cells are simply  in PBS alone and are washed of  
free dye before being treated in full RPMI media without FBS.
We want to be able to make comparative study of the samples and the  
kinetics of ROS generation over time (starting at t=0) so would  
really like to know if anyone has had similar problems and perhaps a  
solution  for a system of assaying ROS generation with low background  
at t=0	:)

look forward to your responses.


Wayne A. C. Harris
Bone Marrow Transplant Laboratory
Immunology and Flow Cytometry Assays Laboratory
Winship Cancer Institute, Emory University
1701 Uppergate Drive
WCI, building C, Rm#4032
Atlanta GA  30322

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