Patch-clamping transfected mammalian cell lines?

Harry Witchel Harry.Witchel at Bristol.ac.Uk
Sun Jan 18 13:41:09 EST 1998


Hi folks --
    I am trying to optimise my transfection of basic mammalian cell
lines with ion channel plasmids for electrophysiology.  Currently I use
CHO cells, lipofectamine, & a vector that is driven by the CMV promoter,
and my results are inconsistent.  I sometimes have problems with
expression (which seems to be due to DNA quality), but I have more
recurrent problems with cells that do not seal well or that are unstable
while patching.
    I am now at the point where I want to decide if I want to switch
cell lines (to COS or HEK 293), buy a new batch of CHO (I got my cells
from "a friend" who may have been culturing them for over a year), or
clean up my transfection protocol in a detailed way.  Currently I follow
the protocol from BRL for use with the lipofectamine.  One difficulty I
spotted immediately was that the transfected cells had to be washed very
thoroughly before electrophysiology, otherwise the cells were incredibly
leaky.  I am looking for any other similar observations.
    One bizarre factoid is that on some days my cells will not seal
well, but if I then trypsin/EDTA treat the CHO cells (using the same
Trypsin/EDTA solution I use to  passage the cells, but for 1 minute
instead of three) and then wash them carefully, the cells are patchable
and they still express the ion channel (as judged by physiology).  I am
not certain what is outside the cells, but it seems that sometimes they
are making some kind of protective layer on the cell surface.
    So, any ideas about:
Which cells to use?
Should I get a new batch of CHO?
What is the goo on the outside of my CHO cells and how to I deal with
it?
Are there any important steps or tricks to make transfected cells highly
patchable?
E-mail responses appreciated.
    Thanks,
        Harry
Harry.Witchel at bristol.ac.uk

--
Harry J. Witchel, Ph.D.
Dept. Physiology
Medical School
Bristol  BS8 1TD
   England

Harry.Witchel at Bristol.ac.uk
http://www.bris.ac.uk/Depts/Physiology/Staff/hw.htm





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