2 replies to this topic

[Myco Rax]

Hi all,
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.

[bob1]

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....pubmed/20072909

[Myco Rax]

Thank you Bob1...
The info was useful.