Why cells are so resistant to G418? - G418 cannot kill cells (Dec/08/2007 )
Dear all, I have such a trouble with G418 selection. I used 3 batches of G418 which were not effective on my untransfected V79 (Chinese hamster cells line) cells with 1000 microgram per ml. i tried 4 to 10 days but no sign of killing was observed. I searched the bioforum on G418. It seems many people have had this problem. I read that it can take from 2 weeks to one month for G418 to kill the nonresistants. On the other hand in this biofofum i read that G418 can kill the activly dividing cells only. but after 2 weeks my cells even in low cell density plates will have an overgrowth. COULD YOU PLEASE HELP. I AM SOOOOOOOO PANIC!!!!!
other people can do this without problme. see http://www.raraf.org/journal/radres12.pdf. are u sure the line you use is original, direct from atcc? There are other selection marker you can use, obviously, if your problem oersists.
I once did a killing curve with a cancer cell line and could not kill the cell with 1.5 mg/ml G418 for over one week. This cell line usually responds to doses of 0.6-0.8 mg/ml. I thought there must have had something wrong with my cells (contamination?) so I revived new cells and obtained normal result. I am not sure whether this is relevant to your problem. Make sure your cells are healthy.
G418 might be inactive. We had got a new G418 solution and it didnt kill our cells. We had to make it from powder and then it worked. So get a new solution or make a fresh one.
if cells are grown too dense or confluent cells are more stable against G418 than as single or small colonized cells; try a lower cell density
-How do you store your G418 stock (tempeature, solvent-powder)?
-Maybe you should check the stabilty of G418 at 37 and in culture media. You should consider G418 stabilty to decide how often to change the media.
-There some cell lines that express some detoxifing proteins and so it is not possible to select them with a particular antibiotic.
I used to have the same problem with caco cells. Finally I gave it up cause it was likely that they expressed that kind of genes.
a few more questions:
Is your G418 bought as a solution or as a powder?
If you made the solution by yourself, did you keep in mind that you have to calculate the concentration based on the active concentration of G418?
Did you filtrate the solution?