I start soft agar transformation assay using NIH 3T3 cells. These cells shall not form any colonies in soft agar assay. In upper layer of agar these cells survived and start to proliferate (they have round shape). But I found these cells in growing numbers on the bottom of the wells (normal NIH 3T3 shape). I prepared agar layers the same day, bottom agar (0,5%) layer ca. 2 hours before pouring upper layer agar (0,3%) with cells. Does anyone have experience if these cells can "sneak" or crawl from top layer to the bottom of the plate and proliferate there (on 6-well plate, under the layer of agar)?
Unlikely. It is possible that your "NIH 3T3" cells are no longer exhibiting the normal characteristics. Therefore, are able to survive in soft agar without attachment and proliferated. Cells at the bottom of the plate are very difficult to explain (because technically it is very difficult to look at cells at the bottom). However, under certain conditions transformed cells could migrate.
Hi Andrzej,
I think what you see are cells growing on the interface between top and bottom agar - maybe you need to adjust your protocoll slightly. It seems that the top-agar is solidifying too slowly giving cells the necessary time to accumulate on top of the bottom layer. Try to adjust the temperature, let the top agar cool a little before you add it to the wells.
You could also try to reduce the number of cells you are using in your experiments.
Good luck with your experiments,
Christian
One thing to keep in mind when working with 3T3 cells is to use low passages and never let them get above 70% confluency before you split them. Above 70% they will will start to aquire mutations and spontaneously transform. They will exhibit anchorage independent growth in soft agar and loss of contact inhibition.
Good Luck!
If you are sure that the cells really are on the bottom of the dish, then it may be that there is a problem with the bottom layer setting - even though you left it quite a while. Does it seem firm before you add the top layer? If not, maybe the agar is a bit old? Try getting new agar? Or you can increase the concentration a bit, some use 0.6%. Or is the top layer too warm and melting or disturbing part of the bottom layer?
A few thoughts.
Cells at the bottom of this cell/agar culture are easy and clear to see, they look as they are growing in normal growing conditions - in medium with FBS, no agar present. And when compare two plates - one with agar and second standard conditions, when I focus on cels on normal plate and change plate for that with agar I see cells sharp, what is evidence that they are in the same position on plate not on the interface between two agar layers. To see cells in above agar layer I have to adjust focus.And I start to see them after 2-3 weeks of culture, so they have a time to sneak/grow from agar layer to the bottom. I guess that they use side walls in between, but I was unable to see it under microscope. I repeated this with higher agar concentrations and with different agar sources (including Noble agar). Top layer can't be too worm, because in that case cell will be dying, and they stay very alive. So I'm assuming these cells have different culture characteristics - the did transform. I made similar experiments with cos-1 and 32D cells, none of these wish to die when grow in suspension in agar. Especially cos-1 made big colonies.
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