Sorting kills cells?

Nebe-Von-Caron, G g.nebe-von-caron at unipath.com
Fri Jun 18 19:05:01 EST 2004


What you can not see you can not sort / prevent from being sorted. I
happily advocate the low scatter threshold to make sure you see
everything in the sample you sort.  I have recently addressed that
problem of coincidence by unseen events mentioning platelets as an
example, but obviously the problem of debris is equally important. If
your frequency of debris below threshold is for example giving you a
flow rate of 3000 events per second (average one per 10 droplets at 30
kHz) you would expect a 10% recovery just by their random appearance in
your sort. At 1000 events per second I would expect coincidence rates of
unlabeled particles to be below 1% when your original mix would be
50/50, still not explaining the 8% of events you see.

>From your pictures it shows that your original debris do cluster below
your FSC threshold whilst the events after sort form a clear cluster
above the threshold supporting your suggestion of cells dying in the
sort process. A splash of PI would have easily confirmed the problem. It
is quite easily possible for such death to occur and people have
described similar problems on this list before. It depends on the cell
line you use, the nozzle size, the pressure, the sheath liquid,
remaining disinfectant.... but should be avoidable as they are likely to
effect cell physiology of the cells in subsequent culture.

I take it you checked pure sheath collected from the system w/o sorting
cells and the impact of exposing cells to your sheath solution to
exclude its interference. 

Good luck

Gerhard


----Original Message-----
From: Ray Hester [mailto:rhester at jaguar1.usouthal.edu] 
Sent: 14 June 2004 20:50
To: cyto-inbox
Subject: Sorting kills cells?


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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