Sorting kills cells?

Ann Atzberger ann.atzberger at talk21.com
Wed Jun 16 08:42:23 EST 2004


Hi Ray,
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.
regards
Ann

Ray Hester <rhester at jaguar1.usouthal.edu> wrote:
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



This attachment - 'GFPSort 001.jpg' - 163.75 KBytes - can be viewed at
http://www.cyto.purdue.edu/MD-parts/7c8a54c60a0e179890ea9930b0404912297dcee8.jpg 


---------------------------------
 ALL-NEW Yahoo! Messenger - sooooo many all-new ways to express yourself 
-------------- next part --------------
HTML attachment scrubbed and removed


More information about the Cytometry mailing list