I'm going to word my problem in a generalized way, since I've seen this come up in several diffent types of experiments: I have examined two cell surface antigens, A and B, using single color (FITC), indirect antibody staining and flow cytometry. The cells are a stable line that is 100% positive for both A and B under all circumstances. I wish to quantitate changes in A and B under various treatments by examining the mean fluorescence intensity of flow samples. For Treatment X, A goes up slightly and B goes down dramatically compared to no treatment (control cultures stained with A and B antibodies). Changes in both A and B are both reproducible and statistically significant. However, the autofluorescence signal (cells stained with buffer without any antibody) also goes up slightly for cells under Treatment X compared to no treatment. (None of the isotype controls are affected under any condition or under any circumstance and are always identical to the autofluorescence signal.) I have the voltage set such that the autofluorescence signal is trivial compared to the signal of either A & B, so I didn't bother subtracting it out. Instead, I tried normalizing all my data to the autofluorescence signal by taking the ratio of Treatment X stained cells (XA or XB) over Treatment X unstained cells (Xauto) and compared this (XA/Xauto or XB/Xauto) to untreated control cells (CA/Cauto or CB/Cauto). By doing this, I get a different interpretation of my results. The increase in A is very similar to the increase in autofluorescence, or maybe even less, so that with the normalization, I find that Treatment X has no effect on A, or possibly a decrease in A, compared to no treatment. The effect of Treatment X on B is even more pronounced after normalization. How do I interpret my results? What is the significance of a change in a cell's autofluorescence? I notice that by changing the voltage on the instrument, the ratios of A/auto and B/auto both remain constant, but does this necessarily justify the normalization procedure to cancel out biological-based changes in a cell's autofluorescence? In this circumstance, it appears justified because the changes in A and auto are directly proportional. Or might this just be coincidental? The proportional changes in A and auto are very similar over both time and concentration of X. Should changes in forward scatter be proportional as well? Thanks, Jim -- James A. Zanghi Dept. of Chemical Engineering Northwestern University Evanston, IL
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