On a six-well culture dish there is one well, which is overcrowded with cells and this happened during a weekend (difference can be observed even without microscope - see picture attached). They were infected with a recombinant baculovirus strain by lipofection and there were the same amount of cells in each well, to begin with. All the other wells are normal, including the mock-infected and the cell control. The abundant cells look absolutely as they should though, they are just like six times more then the ones in all the other wells. 2% of FBS was applied with the medium (no antibiotics). Has anyone ever encountered such phenomenon? If so, what do you think the reason could be?
Hi Judit,
Odd. The first thing that would spring to my mind is that I had mixed up the wells, and that this is the untreated cell control. I'd repeat the transfection to see if I could replicate it.
You say the cells look as they should, is this for uninfected cells or infected cells? Have you generated virus from these cells, or from those that you think should not produce virus?
If you can rule out the simple mistake then you have a interesting phenomenon there that I've never seen before. Does the function of the protein you will be expressing have anything to indicate that it might cause such an effect?
-Richard
Hi Richard,
Thank you for your answer. I have repeated the experiment with the very same setup. Fortunately, I can rule out the chance of mixing up wells with quite a good likelihood; I never use the middle wells as untreated controls, moreover, all the wells were well marked even the possibility of putting the lid on in the wrong way can be excluded as I always mark one edge.
They look like the healthy cells should, however, they are rather tiny (compared to cells in a not-so-super-crowded monolayer. Unfortunately we don't have a camera here, so cannot take a picture of them in the microscope.
The protein expressed should be the capsid protein of a densovirus... To be honest I have no idea if it could have such an effect, but since it is a structural protein my guess would rather go for some roles in cell entry instead of cell multiplication. Nevertheless, you can never rule out this opportunity.
I am quite new to baculoviruses, can they immortalize cells? Is there any chance to check markers on these cells to determine if they are immortalized? (That was my idea at least when I first saw them)
-Judit
Hi Judit,
It will be interesting to see what the repeat shows.
They look like the healthy cells should, however, they are rather tiny...
How much smaller? Uninfected cells will be around 13um in diameter with infected ones around 16um. This is a noticeable size difference but is not great. If the cells you are seeing are much smaller than the uninfected cells then I suspect that you may have a contamination in the well, probably from a yeast, as they can look like very small insect cells and of course double much faster. It might be worth just checking that you haven't got a contamination in your DNA by adding it to some media.
The protein expressed should be the capsid protein of a densovirus...
I would be surprised if that would have an effect, but I've been surprised before :)
can they immortalize cells?
Not that I know of. The virus is a lytic one, and the DNA is extrachromosomal so it would be a very strange occurrence. The insect cells are already pretty much immortal anyway.
Is there any chance to check markers on these cells to determine if they are immortalized?
Don't know.
-Richard
I had the same thing happen. In my case it was yeast contamination!
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