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Measurement of ROS by h2DCFDA


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#1 Myco Rax

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Posted 29 September 2012 - 09:02 AM

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.

#2 bob1

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Posted 29 September 2012 - 12:21 PM

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

#3 Myco Rax

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Posted 30 September 2012 - 10:19 PM

Thank you Bob1...
The info was useful.




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