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Sorted cells rarely 100% pure. However, you can get very very
close. (BTW, you should always always always do a reanalysis of the
sorted cells. It is always worth using 10% of your sort volume to
determine the purity. Remember to wash ethanol and then PBS through
the sample lines before running the sort, because just a few cells left
over in the lines can artefactually affect your purity!).
I typically get 99.3 to 99.8% purity. I always do 2 drop sorts. Dave
Parks has a very good methodology for setting up the optimal sort
conditions; you could ask him for the specifics (Parks@Darwin.stanford.edu).
(Sorry, Dave!).
Best sort purity can be obtained by sorting on a combination of
positive AND negative selectable markers. I.e., to sort CD4 T cells,
ideally you would sort on CD3+, CD4+, AND CD8-. The negative marker is
useful to exclude doublets. We often use a "dump" channel, in which we
put all of our negative markers. Typically this is the PE channel (or
Cy5PE channel), so that we can include PI as a negative marker as well
(and thus get live cells only). Note you could also use a texas red
channel (PI fluoresces in so many places!).
Another useful trick to increase recovery is to perform "vertical"
sorts. I.e., set the deflection so that the main stream is diverted at
an angle (and the aspirator is moved as well. For the BD machines, you
can pull the aspirator out a little, and then wedge it to the side
using a cut yellow pipet tip (thanks Dick Stovel!). The deflection is
set so that the sort stream is vertical). There are two advantages to
this method: (1) the sorted drops have no charge, thus there is no
charge buildup in the tube, and the sorted cells don't "bounce" out
after a while; (2) the vertical orientation of the sort stream results
in a smaller target area with less splatter. Of course, this is only
useful if you are doing single-stream sorts.
mr
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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