A problem with SF9 insect cells

[Anthony Davies]

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.

Anthony Davies
Institute of Virology
Oxford UK