Technical FAQ

Why are my cells not growing well or why are they enlarged and floating?

The cells may have been left too long between passages, and have overgrown, which can result in some floating. They can be sub-cultured like this to produce a fresh healthy culture, but should not be used for making or amplifying viruses. The cells may also be old, i.e. have undergone more than 30 continuous passages since being raised from liquid nitrogen. You will need to raise a fresh batch of low passage number cells from liquid nitrogen. Enlarged, floating cells can also be indicative of virus contamination of stock cells, particularly if the nuclei are enlarged. Always handle stock cells and medium before handling virus. Other problems may include the use of incorrect medium or incubation conditions.

Why has my co-transfection become contaminated?

Your transfer vector DNA may have microbial contamination. Phenol/chloroform the DNA, ethanol-precipitate and re-suspend in sterile TEbuffer. Alternatively, your lipofection reagent or your medium may be contaminated. Sterility test a sample of each by adding to a small volume of culture medium and incubating at 28°C and 37°C for 48 hours. Then monitor for signs of contamination using a phase-contrast microscope or plate out onto bacterial plates.OXFORD EXPRESSION TECHNOLOGIES 57 The flashBAC and control transfer vector DNA provided in the kit has been purified using CsCl density gradients and has been tested for sterility prior to packing.

Why has my co-transfection failed to produce recombinant virus?

Check that the cells are healthy using a phase-contrast microscope and that they did not dry out during the co-transfection procedure. If using cells normally cultivated in serum-supplemented medium ensure that you washthe cells with serum-free medium before using them for a co-transfection. Remember to add the extra 1 ml of appropriate culture medium to the co-transfected cells after incubating them for 5 - 24 h.
In our experience there are two main reasons why co-transfections do not work. The first of these is that the cells are unhealthy and the second is the presence of serum. It is important to be aware that many of the transfection reagents on the market that are advertised as being effective in the presence of serum may not in fact work in insect cells when serum is present. This is because many manufacturers test and optimise only for transfection efficiency in mammalian cell lines. To avoid problems at this stage we strongly recommend that co-transfections are conducted without the presence of serum. Co-transfections may fail if they are carried out in the presence of some commercially available formulations of serum-free medium and we recommend using a less complex media for the intial stages of procedure such as TC100.

Why has my virus amplification failed to produce high titre recombinant virus?

Check the condition and density of your stock insect cells. If the cellsare seeded too dense they will inhibit virus replication. The virus titrefrom the co-transfection may be too low for the volume of cells beinginfected. You will need to leave the infection for longer than 5 days andmonitor virus amplification using a phase-contrast microscope to determinethe optimal harvest time. Conversely, you may have added too much virus tothe culture of cells so that only one round of amplification has occurred.See section 8.2 for more advice.

Why do I not see any plaques on my plaque assay?

Ensure you are using healthy cells and avoid dislodging cells whenreplacing medium. The cells should adhere to tissue OXFORD EXPRESSION TECHNOLOGIES 58culture dishes within 1h after plating, with very few floaters present. Otherwise, discard dishes and obtain fresh cells. Check the cellconcentration was not too high or too low when seeding the dishes. If the cells are seeded too densely then the virus does not replicate properly and plaques will be very small - so small that they can only be seen under the microscope. This is a very common problem with plaque-assays! Seeding cells too thinly will require a longer incubation period to produce confluentmonolayers and also results in large, ill-defined and diffuse plaques. Ensure that the temperature of the agarose overlay was not too high (>37oC) when poured over the cells or that the cells have not been allowed to dry out at any stage. The latter is characterised by large areas of bright pinkstain with a glassy appearance. Remember to add serum-supplemented medium to the agarose overlay when usingcells that require serum and don't forget the 1ml liquid feed overlay when the agarose overlay has set. The virus titre may be too low to be detected on the dilutions that you have plated out (try plating out the lower dilutions too from 10-1 to 10-7). Or the virus titre may be so high that it has lysed all the cells and plaques have merged into each other (plate out higher dilutions). A common error that results in this problem is to forget to change tips when preparing the virus dilutions - so that virus carry-over occurs and gives a false high virus titre. Always prepare freshly diluted Neutral Red for each plaque assay.

My agarose overlay has cracks and/or my plaques look smeared and/or plaques are all located around the edge of the plate. Or my agarose overlay came away from the dish when I inverted them. Why?

If the virus inoculum is not completely removed from the cells beforeadding the agarose overlay, it will interfere with the gelling process andproduce cracks. This may also cause the overlay to fall away when the dishesare inverted! It may also cause the plaques to look smeared by allowing thevirus to spread randomly, rather than being contained within foci of cells.Always seed the cells uniformly in the dish and ensure that the virusinoculum is added to the centre of the dish dropwise to cover the cellsevenly or it can also cause smearing and may result in plaques predominatingaround the edge of the dish.

Why can I not detect expression of my gene?

Were the cells in good condition and in log phase of growth when used forthe infection? If not, the virus may not have been able to replicate and thepolyhedrin gene promoter may not have been activated (see section also 10). Have you titrated the virus to know that the cells have actually beeninfected? If not this is important, as the virus may not have amplified forsome reason (see section also 8.3).Has the virus been stored for some time before use? If so, check the titre(see also section 8.3).Does the control recombinant lacZ virus give good levels of â-galactosidase?If not, you may need to revise your cell culture and cell infectionprotocols.
Is the coding region of the gene downstream of the polyhedrin gene promoterinserted in such a way that the gene's AUG start codon is the first AUG after the promoter sequences? This is important as translation occurs at the first AUG in the mRNA. If you have added tags or other sequences, are they in frame? Have you checked your construct by DNA sequencing? Has the gene transferred from the transfer vector to the baculovirus genome? Check this by extracting DNA from virus-infected cells and analysing by PCR. It is very, very rare that this is a problem. Have you optimised expression conditions - cell line, time to harvest?