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The instrument/reagent negative control would cells with no staining.
On a two-parameter PI (y) vs FITC (x) plot, all of these cells should
appear in the lower left-hand quadrant.
The physiological negative control would be the happiest cells one could
produce stained with both PI and the FITC-annexin. By happiest, I mean
intact, non-apoptotic cells, free from dead cells and cell fragments.
Ideally, all of these cells would also appear in the lower left
quadrant.
The positive control would be stained with both PI and the FITC-annexin,
and treated in a manner know to induce apoptosis IN THE TYPE OF CELLS
BEING EXAMINED. With lymphocytes and fibroblasts, I have found serum
starvation or treatment with dexamethasone to work well. With this
control, as with the samples for testing, viable cells will be in the
lower left, early apoptotic cells will be in the lower right, and late
apoptotic or necrotic cells will be in the upper two quadrants.
I like to use the compenstaion controls, even though I read FITC on FL1
and PI on FL3 on my Coulter XL so that there is a minimal amount of
emission overlap. Cells stined with FITC-annexin only should not apper
in either of the upper quadrants. Likewise, cells stained with PI only
should not appear in either of the right-hand quadrants.
I suppose one could skip these compensation controls if so inclined.
Furthermore, after using this rigorous control scheme a couple of times,
one may find that it is appropriate to cut back to just the
physiological control.
Have fun!
Mark A. Miller
mamiller@wista.wistar.upenn.edu
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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