Hi Juan,
Even though you're intending to "see" PI only in FL2, it fluoresces brightly
in FL3 as well. Both 7AAD and PI are excluded by viable cells, but enter
dead cells and stain their DNA brightly. I'd imagine that the
sub-population that you see is a dead (or dying) one.
How "spontaneous" is the signal that they give? Do you actually have 7AAD in
the tube, if not have you thoroughly removed all traces of 7AAD/PI from
previous runs before adding your cell line? Have you looked at the cells
under a microscope to see if the staining correlates with any morphological
trait ?
I hope this helps,
Ray
Ray Hicks
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> From: "Juan Oliver M.D." <jao7@columbia.edu>
> Date: Wed, 14 Mar 2001 16:22:29 -0800
> To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>
> Subject: PI signal in FL3
>
>
> I have an embryonic pluripotent cell line that has a subpopulation of
> cells
> with a spontaneous strong signal with a FACScalibur in FL3 (7AAD); the
> remarkable finding is that this population of cells increases its signal
> one log in
> FL3 after PI addition (FL2); 10 micoliters of 0.1 mg/ml into ~ 1ml .
> Nobody in our facility can explain it; any suggestions would be
> appreciated. Thanks, Juan Oliver
>
>
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