Well there are
several issues.
First, the source of the 2'7-DCFHDA is important - my
own experience over many years is that the Molecular Probes reagent is the only one to
use - Kodak make it and I tested a couple of lots and they were not
good!
Second, yes -
this reaction
is well known. We published a few papers back whenever -
that discuss some of the reaction issues
(Measurement of
intracellular
fluorescence of
human monocytes relative to oxidative metabolism, Robinson,J.P.; Bruner,L.H.;
Bassoe,C.-F.; Hudson,J.L.; Ward,P.A.; Phan,S.H., J.Leukoc.Biol.43:304-310,
1988)
Third, there
will be a continuous
oxidation taking place in cell systems regardless of the
stimulation you might place your cells under. Under a confocal or fluorescence
microscope, you need to be careful because I believe there is some photooxidation
Finally,
lymphocytes are
not going to produce a whole lot of oxygen radicals - in fact,
you will have a lot of trouble discriminating the background from the stimulated cells
using 2'7-DCFHDA. I suggest that you use a more specific fluorochrome. In fact, I am
not surprised you cannot get a stable response since the background is going to be
significant.
regards
paul
robinson
purdue
university
-----------------------------------
On 20 Nov 2003 at
16:52, Sabyasachi
Biswas wrote:
>
Hi
>
>
Does anyone know
whether 2',7'-dichlorofluorescin diacetate (H2DCFDA)
>
photodecomposes?
We are trying to detect H2O2 generation in lymphocytes but our
>
results are anomalous.
Microscopic examination suggested that H2DCFDA decomposed
>
and fluoresced
when excited with FITC filter in our fluorescence microscope.
>
However we couldn't
find any reference for this phenomenon. Any information
