2 replies to this topic

[Guada]

Hello,
I have been trying to generate Vero cells stably expressing a Golgi protein fused to GFP or a membrane protein fused to GFP. I started 3 months ago with this and until today I do not seem to have any positive colonies! I did different transfections and after 48h I split my cells in the presence of G418 (1mg/ml). My control cells they all die within 10 days but in my transfected cells I see little colonies after this time. When I check my cells in the fuorescence microscope some of them are positive but the colonies are completely heterogeneous. When I split again the cells I loose my few positive cells....
Any idea how to improve this?
Thank you very much!!
Guada

[Akdor]

Is it not too much 1mg/ml of G418? Are you using the lower concentration to kill the controls? It sounds to me that you are using too much Neomycin and your cells are suffering...

[Guada]

Hi! Thank you for your answer. I did a killing curve on my Vero cells and 1mg/ml was the concentration recommended for mammalian cells and that killed all the control cells in around 10 days...when I used 0.5 or 0.8 I saw some cells still alive and growing in my control. But I could try a lower concentration in the transfected cells  to see if I can get more positive cells from the beggining. Thanks!