FITC=//=FITC?
Dr William Smith (williams@ichr.uwa.edu.au)
Thu, 4 Sep 1997 10:41:01 +0800
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
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