Help: Sf9 culture and homogenisation

Tim Green tpg at mrc-lmb.cam.ac.uk
Thu May 25 08:43:48 EST 1995


In article <mbxfd-250595095709 at macfd.biochem.nottingham.ac.uk>,
mbxfd at unicorn.nott.ac.uk (Fergus Doherty) wrote:

> Maybe some of you who use the baculovirus/Sf9 expression system may be able
> to help me with these:
> 
> 1.  In suspension culture the Sf9 cells (non infected) tend to clump, can
> this be prevented?
> 
> 2.  Can you recommend homogenisation methods, I want to recover intact
> organelles, so the homogenisation medium must be iso-osmotic (eg. 0.25M
> sucrose)?  Anyone tried a nitrogen bomb?
> 

In my experience, clumping is best reduced by extensive resuspension when
the cells are passaged.  I find it wise to spin cells down every time I
split them (three times a week).  I then resuspend the pellet by
pippetting up/down 20 times.  Any less than this and the cells tend to
clump.  Cells will also clump if not stirred quickly enough, or
discontinuously (at least 50 rpm).

For the homogenisation, I have no experience of using hypotonic solutions
as I work with total membrane extracts.  I have successfully used a
nitrogen bomb for lysing the cells in low ionic strength buffer (~20mM). 
This worked well with 2-3 litres worth of cells in 250ml, held at 500psi
for 30 minutes.

Hope this helps,

Tim Green



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