Hi collegues, is there anyone who can tell me something about the differences of fluorescence sensitivity on the FACScan and the FACS Vantage SE? I had a badly labelled sample of white blood cells labelled with CD4-CyChrome (PECy5). On the Vantage equipped with a 100um nozzle it was impossible to detect any fluorescence on FL3-channel with a 675/20 BP-filter at 100mW argon laser light. After relabelling a small aliquot of the cells with the same antibody (10ul antibody on 4x10exp6 cells) I could detect a dim fluorescence. Not even a log between the negatives and the positives but good enough for a calculation. I have used antibodies with CyChrome several times before with other cells and never have seen such a bad fluorescence. All the optics were well aligned. But as I said I think the amount of antibody (100ul antibody solution in a 5ml volume of PBS with 2%FCS for 2x10exp8 white blood cells) is not sufficient for proper sorting. Interestingly the CD4-CyChrome bad labelled cells could easily be seen on the Fl3-channel on our old old FACScan. Even with this little amount of antibody there was a clear and beautiful difference between CD4pos. and neg. cells. The relabelled aliquot of cells didn't give any improved signal on the FACScan. Any suggestions? Thanks in advance Best regards Volker Volker Eckstein PhD Dept. of Internal Medicine V Medical school of the university University of Heidelberg GERMANY volker_eckstein@med.uni-heidelberg.de
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