Freezing down intact organelles

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Thu Sep 2 11:04:31 EST 1999



Trond Olav Berg wrote:

> I am working on an in vitro fusion assay, trying to get isolated,
> purified autophagosomes to fuse with lysosomes. It is a lot of work to
> isolate these organelles, and it would of course be a large advantage to
> freeze them down.
>
> I am therefore looking for good procedures for the freezing down of
> intact organelles, of course as gently as possible, so the organelles
> are ready to fuse after they are thawed.
>
> Good protocols are appreciated!
>
> Trond Olav Berg

Hi Trond,
I don´t know about organelles in particular, but I guess that freezing them
down like live cells should work. It is very simple:
* to the suspension of organelles/cells, add DMSO to a final concentration
of 10%
* freeze them to -70degrees SLOWLY. For cells, 1degree or less per minute
is best. You can achieve that with a special  plastic chamber partially
filled with isopropanol (sold e.g. by Nalgene), or by wrapping the
cryovials in thick layers of tissue paper and placing it in a small
styrofoam box.
* after one night at -70, place into liquid nitrogen for long term storage

* for thawing, take out a cryovial and IMMEDIATELY immerse it in warm water
(37degrees) until thawed. Do not allow it thaw slowly.

Hope this helps,
Frank




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