A problem with SF9 insect cells
adavies at vax.oxford.ac.uk
Tue Feb 4 03:47:11 EST 1992
In article <839.298d8ac8 at nphi.fi>, tronni at nphi.fi writes:
> We use a baculovirus expression system with SF9 insect cells.
> I have an obscure problem sometimes when I try to titer my
> virus stocks and search for recombinants.
> I always use 2 million cells per 60 mm Costar plate,
> infect cells with 1-2 ml of baculovirus stock dilution
> (1/10000-1/100000) for one hour and take liquid very
> carefully away before adding TNMFH agar-media (42 Celsius degrees)
> to the plates (10 % FCS).
This cell density sounds rather low. Try using 1.5x10^6 cells per 35 mm dish
for medium sized plaques.
I incubate for 6-7 days in 28 Celsius
This is a *long* time! Much better incubate 3 days at 28C, then neutral red
stain for one hour to visualise plaques. I suspect your cells are often
pretty dead by the end of a week.
> Sometimes plates have a highly fuzzy "background" and no plaques
> can be seen against that background. The whole plate or big
> part of it looks silvery opaque.
Another possibility is that the Sf9s are not in tiptop condition when infected
at the start of the assay. Infection will then be very inefficient, giving a
serious underestimate of the pfu/ml.
> "Background problem" is not persistent but a nuisance anyway.
> Some plates can be OK, even though they have been prepared at the
> same time from the same batch of cells.
> Has anyone any similar experiences?
Anyone who wants a guaranteed (why am I setting myself up like this? :-))
protocol can Email me.
Institute of Virology
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