Sf9 Cells

BAKERK at UK.AC.AFRC.FRIR BAKERK at UK.AC.AFRC.FRIR
Sat Aug 29 08:54:00 EST 1992


>  Hi! I was wondering if anyone out there has experience working with Sf9 cells
>isolated from the ovary of armyworms.
>  I pretty much follow the protocol of Summers and Smith. Using Grace's medium
>supplemented with 10% FCS and the specified concentration of Gentamicin.
>  However, recently my cells begin to die after a few days of incubation.
>Specifically, after reviving the cells from liquid N2 and seeding 2e6 cells in
>a 25cm2 flask, the cells are fine and reaches confluency. I would then pass the
>cells 1 to 3 or 1 to 4 in about 5 days. But appoximately 2 days after the 1st
>passage, the cells would begin to die. They would float, stop dividing, and I
>think that I can see holes in them (some bright spots?). Don't think it's
>contamination by baculovirus though that's what I'm growing the cells for?
>Has anyone experienced this? Any suggestions?
>  Are these cells susceptible to mycoplasma contamination? The gentamicin that
>I'm using is from BRL and the maker describes as effective against mycoplasma.
>  I've also heard that a "new?" medium called Sf900 is better. Any comments?
>   How many passages can I achieve with these cells continously
>  Right now, I plan on purchasing new cells from INV.
>  I would appreciate any comments or suggestions.
>  Thanks in advance.

>a52041 at sakura.kudpc.kyoto-u.ac.jp
>Michael Cheng

The 'holes' that you can see are almost definitely polyhedra, indicating that 
your cells are infected with polyhedrin-positive baculovirus. Throw out all of 
your opened bottles of medium, serum, etc., buy or thaw out new cells and start 
again.
Better luck next time.
Cheers,

Ken.
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Dr Ken Baker                              JANET : UK.AC.AFRC.FRIR::BAKERK
Department of Protein Engineering      INTERNET :  BAKERK at FRIR.AFRC.AC.UK
AFRC Institute of Food Research             TEL :        (+44) 734 357139
Reading                                     FAX :        (+44) 734 267917
Berks RG6 2EF
UK                          (AFRC = Agricultural & Food Research Council)
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