The MolProbes catalog lists the conjugate as "fluorescein labeled
dextran", not FITC. My guess is they used FSX, fluorescein-x
succinimidyl ester, which has a longer wavelength emission and hence
requires more compensation.
Calman
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From: Dr William Smith
Sent: Wednesday, September 3, 1997 10:41 PM
To: cyto-inbox
Subject: FITC=//=FITC?
Excuse my ignorance, there is probably a simple answer to this but-
Are
FITCs not all equal?
Using most commercial and in-house FITC conjugated antibodies, I find
that
the overlap into the FL2 channel on our Coulter XL can be compensated
with
a 16-20% subtraction of the FL1 signal, which is of course normal.
However, using FITC-dextran (Mol. Probes) there is a much greater
overlap
into FL2 which cannot be satisfactorily compensated out. This is not
due to
relative brightness, which is often greater in the conjugated
antibodies.
Furthermore, recently I tested a bottle of commercial FITC-GAM which I
was
given, from the back of someone's fridge, expiry date several years
ago. It
seemed to stain the cells well, but again, I noticed a huge signal in
FL2
which wasn't compensated out even with a 70% subtraction. Using this
marker
alone, ie. no PE, and 20% compensation, all FITC positive cells were
also
positive in FL2. With 70% compensation, the majority of FITC labelled
cells
were on the baseline but many were still showing positive in FL2.
Is there an explanation for this? FITC isomers, someone has told me?
______________________________________________
Dr William Smith
Institute for Child Health Research
(Company Limited by Guarantee ACN 009 278 755)
Subiaco, Western Australia, 6008
PO Box 855 West Perth WA 6872
Ph 61 8 9340 8792/8388, Fax 61 8 9388 3414
email williams@ichr.uwa.edu.au
______________________________________________