NIH 3T3 Stable Line Transfection - (Sep/29/2011 )

Hello,

This is my first post, so I'll give some background.

I have been trying to establish a stable transfected cell line of NIH 3T3. At first it appearred successful, I used G418S antibiotic for cell selection. A week post transfection and selection and a very small black rod shaped substance began attaching to the otherwise healthy looking cells. The rods appeared like a beard around the cells. At first I believed it to be cell waste and the normal cell death that occurs with selection however after an additional week the black rods are multiplying and sufocating the remaining healthy cells. The cells no longer reproduce and do not adhere as they should. What ever this is, it is not killed by pen/ strep or the G418S. Also, this substance does not change the media or completely kill the cells. I am now at week 4 and don't want to give up on these cells. Does any one have any ideas ?

There seem to be some bacteria, which are commonly present in waterbaths, that cause black dots in cell cultures. There is a pretty good chance that you have one of these sorts of things.

If you really really need to keep these cells - try some different antibiotics, and combinations of antibiotics.