Has anybody generated stable cell lines using HepG2 cells? - Stable cell lines (Jul/14/2005 )

Hi,

I am trying to generate my first stable cell line using Hepg2 cells. I am worried that this particular cell line will be extra difficult as the cells do not like being too diluted in normal tissue culture practise so will not survive well as single colonies

has anybody used these before and if so do you have any tips?

Crystal baker

Hi,

I've done this ...and its working well, you get great colonies and they are easy to pick....so....GOGOGOGO :-)



Greetz


K_R

i
i've got problems regarding G418 selection in this cell line... It's a long time to get good selection. So i've tried to make clones from culture in an increased conc. of G418. And it worked...
fred

I've also done this. Using a concentration of 1ug/ul of G418 I had good selection in 2-3 weeks.

Hi,

You previusly replied that you had succesfully created stable cell lines in Hepg2 cells. Unfortunatly I am still not having much luck, My antibitic selection works well but I am not getting any surviving colonies even after 3 weeks. I have done it twice now. I am currently using the Promega TFX reagent which works well with my luciferase transient based assays.

Please can you give me any adivice? Did you linearise your plasmid? what transfection method did you use?


many thanks

Crystal

hi
i use lipofectamine without antibiotic in the medium, and let stay the mixture for 5h. Then complete by a 2Xserum medium.
For DNA : i use a ratio DNA/lipoectamine by 1:6 and use 10µg DNA for a 15cm plate. I don't linearize. I wait the thrid day to start selection.
fred