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>I then sort out the top 15% or so of the brightest FITC labeled cells,
I'm assuming you mean the green fluorescent GFP labeled cells
> out
>of the right arm of the gaussian (no second peak is distinct for the
>positives, but rather what we see is a continuum of increasingly brighter
>cells). Everything looks great for the sort (I check a sample under the
>fluorescent scope, number of sorted events are accurate, etc.) until I run
>a portion of the sorted sample back through to check for purity. I get
>about an 80:20 ratio of positive to negative cells, which fall out in 2
>distinct peaks, scatter is still perfectly intact.
>
When cell populations have broad scatter distributions, scatter is fairly
reliable for discriminating cells from small debris, but unreliable for
discriminating single cells from aggregates. In my experience, based on
adding Hoechst dyes to samples, there can be a substantial number of
aggregates in a sample when you wouldn't expect them to be there. They
won't be eliminated by coincidence circuitry. An aggregate will be brighter
than the average cell, and the upper tail of your fluorescence distribution
is likely to contain some aggregates, some of which will be composed of a
highly fluorescent cell and a dimmer one. If they disaggregate between the
time you sort and the time you resort, this will give you a distribution
with bright and dim peaks. I'd say this is the most likely explanation of
your results.
-Howard
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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;
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