Richard, if it's any consolation HEK 293 cells are the pain of my life
too. Unfortunately they are rather easy to transfect and respond well in
calcium assays, etc. so seem to have become a standard in the
pharmaceutical industry. Nobody thinks about the poor person trying to
create the cell lines. Basically HEK 293 cells do not like to be alone in
the well, so many clones do not start growing. Could I suggest if you are
using a selection reagent in your medium to isolate cells expressing your
receptors, that you pre-seed your wells with some wild-type HEK 293 cells
that are sensitive to your selection agent, geneticin, hygromycin,
whatever. They should survive long enough to provide the platform for
your sorted HEK 293 cells to grow. I have used this technique for other
cell types but not HEK 293. If you do get it to work, could you share
your findings?
Hope that's helpful.
Simon
GlaxoSmithKline R&D UK
"Hastings, Richard C" <richard.hastings@astrazeneca.com>
18-Dec-2002 15:57
To: "Cytometry Mailing List"
cc:
Subject: Sorting HEK 293 and establishing clonal cell lines
Hi,
This message is related to the CHO cell sorting thread. I perform many
sorts
with HEK 293 and CHO transfected with different receptors. I generally
perform single cell deposition into each well of 96 well plates to
establish
clonal cell lines. My problem is getting these single cell clones to grow
especially with the HEK 293 (I have much better success generating CHO
lines). I have tried all sorts of little tricks to get these cells to grow
such as using conditioned media in the wells, and using collagen- or
poly-D-lysine-coated plates. My success rate with single cell deposition
with HEK 293 is quite poor, I am estimating its probably below 1%.
Meaning,
I am lucky to generate one clone per 96 well plate. Of course, success
depends quite a bit on the gene transfected into the HEK 293 and the
health
of the cells at the time of sorting.
Does anybody know of any conditions that would increase the number of
clones
I could generate using HEK 293?
Thanks, Rich
Richard C. Hastings
Associate Scientist
Target Expression/LDD
1800 Concord Pike
Wilmington, DE 19803
302-886-2452
richard.hastings@astrazeneca.com
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