tricks to increase retroviral titer?

Aawara Chowdhury via methods%40net.bio.net (by aawara from FEMA-trailer.org)
Mon May 28 11:07:07 EST 2007


In <1180363688.517662.228520 from q75g2000hsh.googlegroups.com>,
 Antonio <Antonio.Sarikas from gmail.com> wrote:

> Thanks DK, but what do you pellet? The virus ist secreted into the
> supernatant which I then collect at day 2 and 3 posttransfection.
> Ultracentrifugation of the supernatant might be problematic since the
> retroviral glycocalix is sensitve to shearstress.

DK is correct, you can definitely concentrate retroviral supes by
ultracentrifugation.  I did this routinely with retroviruses as a 
graduate student, and now my lab does it with amphotropic vectors 
and pantropic vectors.

Here's how we do it -

a) Layer 33 ml cell supernatant on 5 ml 20% sucrose in an SW 28 tube.
b) Spin at 26,000 rpm for 2 hours at 5oC.  Make sure the rotor, centrifuge
   and buckets are pre-chilled (we leave the rotor and buckets in the
   cold room).
c) You will see a pellet at the bottom of the tube.  Suck the 
   supernatant off.  Resuspend the pellet in 0.5 ml of PBS (or complete
   media - which is what we use).
d) Leave the resuspend pellet O/N at 4oC, and then either freeze at
   -80oC or use.

AC
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