growing cells under agarose
klenchin at facstaff.wisc.edu
Tue Dec 11 13:57:43 EST 2001
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.
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