Viability of sorted cells

Jaume Comas jcomas at sct.ub.es
Wed Jul 7 04:36:52 EST 2004


Dear flow cytometry people,

A problem with the viability of sorted cells was detected in our lab. We have been asked
to sort a
fibroblast cell line, IOT 1/2. Cells have been transfected using fugene, and GFP
expressing cells
(about 15%)and non-expressing cells were sorted into 15-ml falcon tubes filled with
medium (5 ml)
and maintained in ice. Cells before sorting had a clear FS/SS pattern, and viability
using PI was
about 95-98%. The instrument used was a MoFlo; laser is a Coherent Enterprise operated at
150 mW
at 488nm. In the first attempts, a 90-micron tip at 20 PSI was used. A commercial PBS
from
DakoCytomation was used as sheath fluid.

The problem was detected with sorted cells: both positive and negative tubes showed few
cells with
a low FS signal when reanalysed, and viability using PI was lower than 15-20%. Then we
changed to
a 200-micron tip at 5 PSI, but number and viability of sorted cells remained very low.
Non-transfected cells showed the same behaviour when sorted.

I wonder if it is a specific behaviour of this cell line, as we have not observed
viability
problems when sorting other cells at these conditions. Any idea about it will be of great
help.

Thanks in advance,
Jaume



-- 
Jaume Comas
Citometria - Serveis CientíficoTècnics.
Universitat de Barcelona.
tel. 93.403.46.54
fax. 93.403.72.66






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