Re growing cells under agarose
wolfsc at ibms.sinica.edu.tw
Tue Dec 11 20:36:00 EST 2001
As you may imagine, it's the continued story of growing cells on cellophane.
They didn't like it and preferred to hide beneath the sheets (if apoptosis was
not happening quicker). They also didn't like it when I soaked the membranes
in diluted bleach or hydrogen peroxide in order to kill possible moulds.
I found a nice plaque lift assay procedure in a recent nature biotechnology
paper (Koller-D et al, www.cytos.com, see DELPHI), but as usual in nature
journals, not giving a lot of experimental details. Although I never have been
working with baculo, I started to devise a procedure similar to the
suggestions that were made by people in the NG. Now I have enough information
to pour the stuff onto the cells. Lets see what comes out. I am just curious
if normal agarose will let diffuse a 270kD tetramer to the membrane...
PS Did you remove your antispam address?
> wolfsc at ibms.sinica.edu.tw ("Wolfgang Schechinger") wrote:
> > hi all,
> > I'd like to cover fibroblasts with agarose or another protein free jelly.
> > Is there anyone aware of how to pour liquid agarose (or equivalent) onto
> > the cells without boiling them?
> It's a standard technique in baculovirus plaque purification.
> Exact procedure is below, couple points: Low melting agarose can be
> used but is not good. Not solid enough and tends to start sliding in
> the dish when there is an excess of liquid. Don't be afraid to
> fry cells - agarose cools down fast enough. Even insect cells that
> are getting heat shocked at >32C are fine. Purity of agarose can be
> important. Agarose I use is Seakem ME from FMC.
> Make 1.5X medium, autoclave 2% agarose in H2O, keep both in water
> bath at 52-55C, quickly dilute agarose to 0.5% final (can keep
> at rt for a while as it does not solidify immediately, I simply
> hold the bottle on a piece of styrofoam), aspirate medium from
> dish(es) removing as much liquid as possible, overlay 4 ml per 60 mm
> dish (I imagine that means 10-11 ml in 100 mm dish), let cool and
> dry with the lid open. If you use LMP agarose, it should be higher %.
> Depending on what you need it for, there an alternative:
> methycellulose gels used to be a standard technique for easy selection
> of cell clones that grow in suspension. The gels are really snotty
> but completely prevent clones from swimming and cell separation.
> Good luck,
Dr. Wolfgang Schechinger
Institute of Biomedical Sciences
Academia Sinica, Taipei, Taiwan R.o.C.
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