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