Hi All,

I am starting to do some soft agar assays using NIH/3T3 cells.

As positive control I used NIH transfected with RasG12V plasmid.

Before placing my cell on the agar gel I noticed (twice and using electroporation or fugene6) that the cells started to develop vesicles (tiny holes inside the cells, bright)...

*Have you ever seen this before? Apparently in some cell lines, the expression of this protein induces Methuosis (a type of cell death Methuosis. Please see the link for "Nonapoptotic Cell Death Associated with Vacuolization of Macropinosome and Endosome Compartments, William A. MaltesexWilliam A. Maltese, American Jounal of Pathology"

*On an agar gel, I couldn't see big colonies forming (8-9 days). How long do you used to wait to see colonies?

*I should use in parallel other cell lines just to be sure that it is not my agar itself has issues. Unfortunately I only have non adherent cell lines (HT-3 cells). I was wondering if I could use this cell as positive control in my agar assay?

In advance thanks a lot for all the advices and information you could provide me.

Have a nice day,

Alice