Measurement of ROS by h2DCFDA - (Sep/29/2012 )
I want to measure the intracellular ROS levels in THP-1 derived macrophages cell line. The test substance is a class 2 pathogen. I want to assess levels of ROS after infecting the macrophages with the pathogen by FACS or confocal. Can anyone suggest me a protocol wherein I can fix my cells, which inactivates pathogen, for FACS or confocal expts? My discussion with people who worked with DCFDA under non-pathogenic conditions suggested me not to fix the cells. But i cannot proceed without inactivating my pathgen before FACS or Confocal, because these instrument facilities at our place are centralised and everyone uses them. Please do suggest me a alternative.
Thanks in advance.
You will need to use the DCFDA before fixing as the fixing will inactivate the intracellular esterases. You should then be able to fix for FACS,
See here: http://www.ncbi.nlm.nih.gov/pubmed/20072909
Thank you Bob1...
The info was useful.