Paul,
If you can use trypsin and it doesn't strip your epitope, try washing
cells with PBS and then use the smallest volume of trypsin at the lowest
concentration you can get away with. I typically use 3ml of trypsin per
T75 flask. The thin film of liquid is enough to detach the CHO cells with
incubation at 37oC. Don't quench with medium containing serum but
immediately dilute trypsin with PBS and then spin down gently. Whilst I
do get some clumps, the majority are single cell suspensions.
Hope that's helpful.
Simon
Simon Rice
GlaxoSmithKline R&D UK
"PAUL HALLBERG" <Paul.Hallberg@mail.tju.edu>
11-Dec-2002 21:38
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
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:26:31 EST