From: Guenter Valet (valet@biochem.mpg.de)
Date: Mon Nov 24 2003 - 04:45:03 EST
2',7'-dichlorofluorescin diacetate (DCFH-DA) activates
under light (Eur.J.Cell Biol 1987; 43:128-33) similarly
as dihydrorhodamine123 (DHR123) or hydroethidine
(HE, J.Leuk.Biol 1990; 47:440-8). DCFH-DA furthermore
hydolyzes slowly and gradually autoactivates like DHR123
or HE when kept in DMSO or in aqueous solutions for
several days or weeks.
As a consequence, stock solutions should be prepared
with the less hygroscopic dimethylformamide (DMF)
and be stored at 4C in a dark place or long term frozen in
liquid nitrogen. Final DMF concentrations in assays should
not be higher than 0.2% for DCFH-DA and not higher than
0.07% for DHR123 since DMF like DMSO are free radical
catching compounds. No electrons are provided by DCFH,
DHR123 or HE molecules at higher DMF and DMSO concentrations
in the presence of ROS, so no cellular fluorescence is generated.
Dead cells should always be counterstained with PI in the three
kinds of assays. Intermediate dilutions of reagent stock
solutions should be freshly prepared just prior to the
cellular experiments and be kept in the dark until the flow
cytometric or microscopic measurements.
DHR123 is more resistant to photoactivation than
DCFH-DA. Following activation to fluorescent DCF, RH123 and
ethidium bromide (EB), all three compounds photobleach
under the microscope relatively quickly. As a consequence
morphologicl fluorescence localisation is more reliably
done with scanning microscopes than with large field
illumination microscopes.
For further details, check:
http://www.biochem.mpg.de/valet/valmeth3.html
Best regards
G.Valet
web: http://www.biochem.mpg.de/valet/cellbio.html
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