Karim: >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 (1) is correct. Although you really ought to have a completely "unstained" control to make sure what autofluorescence is, that your binding is not being caused by other factors (like Fc binding). Nonetheless, that wouldn't change the interpretation that there is no difference and the cells are negative, it would only aid in understanding what's going on. >Another way to state the question: are fluorochrome fluorescence (be it >Ag-specific or aspecific binding) and autofluorescence additive signals >for the same cell? Yes, they are. You might want to consider using Autofluorescence Compensation to reduce the contribution of autofluorescence in your channel of interest. To do this, you must have another fluroescence channel that is "unused", and in which AF correlates well with AF in your measurement channel. Then you can use software or hardware to compensate out some of the autofluorescence. Since this compensation will not reduce specific signal from the fluorochrome, you achieve much greater sensitivity, and can sometimes detect even low level signals against a high AF background. For more information, see papers in 1986 or 1987 in Cytometry on AF correction by spillover correction. One is Roederer & Murphy, the other is Alberti, Parks, & Herzenberg. mr
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