In my experience all FITCs (and PEs) are equal, however all fluoresceins are not equal. 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 ---------- 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 ______________________________________________
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