Sorting kills cells?
ann.atzberger at talk21.com
Wed Jun 16 08:42:23 EST 2004
The question is:
is the debris you see in the sorted sample leftover debris from the original sample? That
is what I am inclined to think as from experience I never seem to be able to get rid 100%
of the original debris/dead cells in a sample, the higher the original debris content the
more you get in the sorted sample.
Ray Hester <rhester at jaguar1.usouthal.edu> wrote:
We recently sorted GFP-positive cells for an investigator and
re-analysis showed what appeared to be, by light scatter, a substantial
number (8 %) of dead cells among the sorted cells.
My question, is it possible that mechanics of the sort (this was a
relatively slow sort - 1,000 cells/sec - through a 70 u nozzle tip)
could result in this number of dead cells that would be displayed as
dead cells by light scatter after being sorted (re-analysis was done
immediately after the sort)? We have a FACSVantage SE and use 200 mW of
488 light to excite the GFP.
In the accompanying attachment, the upper dot plot (061004.003)
represents the unsorted sample, R1 = the sort gate, and R2, what I would
consider viable cells by light scatter. Cells to the left of the R2
gate, I would usually consider non-viable.
The lower dot plot represents re-analysis of the sorted cells. If one
could consider only the cells within the R2 gate, the sort purity would
approach 97% (8903/9147). However, if you consider all of the cells in
the dot plot, the sort purity is, at best, 89% (8903/10,000) - and
perhaps less, since there are additional events off scale to the left.,
and some of these could be cells.
Obviously there are a number of factors to consider, e.g., cell type,
condition of cells prior to being sorted, etc. The investigator reports
the sorted cells, when re-plated, are growing well so it doesn't seem as
if the dead cells (again, by light scatter) seen in the
analysis-after-sorting dot plot are indicative of more dead cells to
follow at some time post- sort.
Nevertheless, I wondered if this type of result (5 to 10% post-sort
dead cells) was a common occurence.
Thanks for any thoughts.
Univ. of South Alabama
rhester at jaguar1.usouthal.edu
This attachment - 'GFPSort 001.jpg' - 163.75 KBytes - can be viewed at
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