Sorting kills cells?

Simon Monard smonard at trudeauinstitute.org
Wed Jun 16 07:50:08 EST 2004


Hi Ray
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.

best


Simon Monard
FACS Lab Manager
Trudeau Institute
154 Algonquin Avenue
Saranac Lake
NY 12983
ph 518 891 3080

>>> Ray Hester <rhester at jaguar1.usouthal.edu> - 6/14/04 3:50 PM >>>
Hi,

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.

Ray Hester
Univ. of South Alabama
rhester at jaguar1.usouthal.edu








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