WHAT CONC. OF DCFDA FOR FLOW CYT.??

From: SHAILAJA KALYANKRISHNA (SKALYANKRISH@utmem1.utmem.edu)
Date: Wed May 19 1999 - 18:52:07 EST


Hello flow cyt. users,

I intend to measure intracellular H2O2 in vascular smooth muscle cells using
H2DCFDA by flow cytometry.

QUESTION 1:

 Does anyone know what conc. (nM or microM) of DCFDA the cells should be loaded
with? I am not fixing the cells and I am carrying them to the flow cytometry
facility (10 minutes walk), so I expect some leakage of the dye from the cells.

I read that DHR123 is more sensitive, but I wish to measure net intracellular
H2O2, rather than mitochondrial H2O2, hence the choice of H2DCFDA.

QUESTION 2:

Some researchers use fluorescence confocal microscopy for the same purpose? 
Are confocal and flow cyt. equally sensitive and accurate for this purpose?

Thanks in advance,
Kalyankrishna S.
SKALYANKRISH@UTMEM1.UTMEM.EDU



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