freezing retrovirus supernatant

[Wolfgang Schechinger]

Hi all,

a quick question, a quick answer is desired. I have some supernatant of
retrovirus producing cells). Is it possible to simply freeze the supernatant or
should I concentrate the virus (how? - this is another question) and add 50%
glyzerol.

Any ideas, recommendations ?

Wo
[tiss mezzage wahs broduceRd using TYPO GENERATOR zoffwer]
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Dr. Wolfgang Schechinger
Lab N233 (c/o Dr. Steve Roffler)
Institute of Biomedical Sciences
Academia Sinica
128 Yen-Chio Yuan Rd. Sec.2
Taipei 115
Taiwan R.O.C.
Tel +886-2-2789-9152
Fax +886-2-2782-9142
Mobile +886-925-136893
e mail wolfsc at ibms dot sinica dot edu dot tw
-----

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[Dr. Duncan Clark]

In article <3B9D25A0.19299.22FD7E8@localhost>, the eminent Wolfgang
Schechinger at messy. wrote

>Is it possible to simply freeze the supernatant or
>should I concentrate the virus (how? - this is another question)

Not sure for mammalian virus, but 10% PEG6000-8000 with 0.5M NaCl (or is
it 1M) is pretty standard fort M13's and lambda etc.

Duncan
--
The problem with the gene pool is that there is no lifeguard.

Duncan Clark
GeneSys Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382

[Victor L.]

It all depends on the envelope. In my hands ecotropic virus can
withstand one freezing (better-flash in liqN2) and thawing at 37^C
after storage at -80^C; you don't need to add anything. Even so, the
titer drops ~4 fold. With VSVg pseudotyped titer stays pretty much the
same after one round.

Unlrafiltration helps but concentrating media proteins makes the final
product quite toxic <why?> to target cells and ecotropic virus was
destroyed <?> since the titer increase was insubstantial.

Your milage may vary....

Victor

On 10 Sep 2001 13:42:19 +0100, wol...@ibms.sinica.edu.tw ("Wolfgang

[Ashok Aiyar]

On 10 Sep 2001 13:42:19 +0100,

"Wolfgang Schechinger" (wol...@ibms.sinica.edu.tw) wrote:
> Hi all,
>
> a quick question, a quick answer is desired. I have some supernatant of
> retrovirus producing cells). Is it possible to simply freeze the supernatant or
> should I concentrate the virus (how? - this is another question) and add 50%
> glyzerol.

Depends on the envelope. Pantropic virions pseudotyped with VSV-G
appear to withstand 1 - 2 rounds of freeze-thaw with no significant
loss of infectivity (as measured by GFP expression after infection).

Viruses with the amphotropic envelope are more susceptible to
freeze-thaw.

We don't add any glycerol. Just flash freeze in a dry-ice/ethanol
bath or liquid nitrogen. Quick thaw at 37oC. Stocks frozen
slowly (1oC/min) at -80oC lose infectivity more rapidly.

Ashok

--
Ashok Aiyar a-a...@northwestern.edu
Department of Microbiology-Immunology office: (312) 503-2524
303 E. Chicago Avenue, WARD 4-123 lab: (312) 503-2542
Northwestern University, Chicago, IL 60611 fax: (312) 503-1339