I have used DCFH-DA in primary microglial (which
express iNOS after LPS + interferon treatment) to
determine peroxynitrite formation. In our hand
this dye worked quite well, although I can confirm
some variability when loading endothelia cells.

The dye was dissolved in DMSO (5mM) and I used
between 10-50 microM final concentration. Although
my knowledge is founded on 'impressions' rather
than science, I strongly recommend to load the
cultures with the DMEM (or whatever else) medium
containing 10 % FCS for 45 min. One washing step
is more than enough.

I tried CM-DCF once and decided to trash it. The
image was blurred and it was difficult to
distinguish cells.

One cautionary note: DCF-H and its derivatives are
highly sensitive to photo-oxidation. I my view it
is impossible to use non-laser excitation like
Hg-lamps, and one should attenuate the laser as
much as possible.

Heiko Possel










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Heiko Possel
Institute for Medical Neurobiology

Leipziger Straße 44

39120 Magdeburg

Telefon 0391-67-14362
Fax     0391-67-14365