Hi Karim By autoflourescence are you meaning non-specific binding of your isotype control? How do totally unlabelled cells look (this is real autofluorescence)? If your isotype control is brighter than your unstained autofluorescence control then your problem is non-specific binding and you have to deal with that. Are you doing direct or indirect staining? If direct then it could be a Fc receptor problem, pre-incubating with mouse serum might help. If you are doing indirect staining then make sure your second layer isn't reacting with something on the cell surface. If your specific antibody (antibody X) and unlabelled cell control (with no antibody at all) are overlayed then your cells may be negative for marker X or it may be obscured by the high autoflourescence for the same reason you can't see stars in the daytime. If this is the case you could try reading your marker in a red channel where autofluorecence is less. Hope this helps some. Simon Monard FACS Lab Manager Trudeau Institute Saranac Lake NY12983 Ph 518 891 3080 X352 >>> Karim Vermaelen <Karim.Vermaelen@rug.ac.be> - 10/20/2000 4:27 AM >>> Hi everybody, Here's maybe a tricky one: Suppose you got highly autofluorescent cells and the isotype control is in the 2nd-3rd decade. The same isotype control in low autofluorescent cells is under the 1st decade. Now suppose the signal for marker X on these high autofluorescent cells is also in 2nd-3rd decade, and exactly overlaps the isotype control. What's the correct interpretation of these results? 1) there's no difference between isotype control and marker X, so the cells are negative for marker X or 2) marker X is in the 2nd-3rd decade, so it's definitely positive regardless of isotype control background Another way to state the question: are fluorochrome fluorescence (be it Ag-specific or aspecific binding) and autofluorescence additive signals for the same cell? TX a lot for any clue! Karim Pulmonary Immunobiology Ghent University Hosp.
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