H2DCFDA and fixation of cells before flow cytometry analysis - (Feb/13/2014 )

Hello,

I want to measure ROS (H2O2 actually) production on my cells by flow cytometry.

My problem is that every sample should have THE SAME INCUBATION TIME of the probe, since the fluorescence increases over time.

So if I incubate 30 min, and then launch the flow cytometer, and that each reading takes 2min/well on a 96-well plate for example, therefore the last well will be incubated with the probe more time than the first one... and the results are not interpratable. For this reason, i was wondering if someone knows an alternative... I was thinking to fix the cells before starting the reading, so i expect to block the reaction from living cells but i am not sure if it is a recommandable technique and that the fluorescence will be stable over time (over 2h for instance).

Thank you in advance for any suggestion,

Sincerly,

No clues?

sure? :)

I'm no expert in this sort of thing, but fixing will kill the cells which would prevent them from producing the ROS.  I suspect that these dyes are designed for reading in a fluorescent plate reader, which would mean that all wells would be read within a few seconds of each-other.  FACS may not be a viable alternative.

Fluorescence may be stable over time, but this depends on the dye and the storage conditions.  Fixing at your end point may quench the dye fluorescence, but this depends on the dye again.

thank you for responding, it may be a good option to try, i'll post a message as soon as i've tried.