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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