Sorting kills cells?

Ray Hester rhester at jaguar1.usouthal.edu
Mon Jun 14 14:50:24 EST 2004


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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