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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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web