Paul,
I have a few comments/suggestions. I've sorted CHOs successfully in the
past. This is what is required. (I'm going to provide all the details so
don't be offended if I mention something that is a no-brainer.)
1. Don't overgrow the cells. If the cells become confluent, they come off
in one big sheet when trypsinized and you'll have difficulty getting a
single-cell suspension.
2. Remove the medium and then add Ca/Mg-free PBS. Add just enough to
cover the cells. Gently rock the flask back and forth a little to wash
the cells then let them sit for no more than 5 minutes. Take the PBS off
and save it as some cells may have already come off. (Assuming that you
are interested in the cells that may have been lightly attached. For
instance, in the case of apoptosis assays these cells that come off easily
may be the apoptotic cells.)
3. Add the trypsin/EDTA solution. Once again, add just enough to cover
the cells. Gently rock the flask again and let it sit at RT for one
minute to two minutes. Check to see if the cells are starting to lift
with a dissection scope. If they are starting to lift, give the
flask/plate a sharp smack on the benchtop. Then look under the scope
again. This should dislodge the majority of the cells. Don't ever leave
the trypsin/EDTA solution on for more than 2 minutes. In my hands, more
than 2 minutes of trypsinization lysed some cells. That's when you really
get big clumps.
Additional/optional steps:
4. If you're having a lot of trouble getting the cells to come loose and
some cell lysis is occurring a little DNAse in the buffer can help prevent
clumping.
5. A different style plate/flask (coating?) may facilitate cell harvest.
6. If you have the capability, and it doesn't affect your experimental
design, you could culture the CHO in suspension with a rotate. (In which
case you can ignore all the previous suggestions!)
I almost forgot. Make sure you use a big enough nozzle when you do the
sorting, since the CHOs can get quite large. I think a 70uM nozzle will
be too small. 100uM is a better way to go.
David McFarland
GlaxoSmithKline
----- Forwarded by David C McFarland/DEV/PHRD/SB_PLC on 13-Dec-2002 08:47
-----
"PAUL HALLBERG" <Paul.Hallberg@mail.tju.edu>
11-Dec-2002 16:38
To: "Cytometry Mailing List"
cc:
Subject: Sorting CHO cells?
I would appreciated any suggestions to facilitate sorting CHO cells which
express VE-cadherin. These cells tend to aggregate making sorting
difficult.
Vortexing seems to increase aggregation. These adherent cells are removed
from the culture flask using trypsin("Cell Stripper"). I try to keep the
cell concentration low which helps somewhat, but is not ideal. Is any
buffer
available that will not destroy VE-cadherin?
Thanks in advance,
Paul L. Hallberg
Flow Cytometry Manager
KCC CORE Flow Cytometry Facility
Thomas Jefferson University
215-503-4556
215-923-0249(fax)
Paul.Hallberg@mail.tju.edu
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