Sorting kills cells?
smonard at trudeauinstitute.org
Wed Jun 16 07:50:08 EST 2004
If its dead cells you should increase the FSC gain to get the whole dead population
above the theshold. Using a combination of forward and side scatter would demonstrate
better whether its debris or dead cells. You would do better to increase the FSC gain and
set a fairly tight gate on FSC and SSC. The "dead cells" you see in the post sort sample
could be stuff that was just below theshold in your pre-sort sample and not de novo dead
cells at all. The rate at which you pass cells through the instrument isn't going to make
any difference to viability. Its the sheath pressure and nozzle size which will
contribute to shear forces that break cells. Increasing the sample rate may increase the
incidence of cells bumping into each other but thats all.
FACS Lab Manager
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>>> Ray Hester <rhester at jaguar1.usouthal.edu> - 6/14/04 3:50 PM >>>
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
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