Hi guys,

I am doing G418 death curve experiments on untransfected TSA201 and HEK293 cell line for my stable transfection. I try G418 from 0.4 to 2mg/ml. On day 6 of G418 treatment, none of the G418 concentration killed cells. On day 11, 1mg/ml G418 or less seemed not to affect cell growth, 1.6mg/ml G418 killed about 1/4 of the cells, and 2mg/ml G418 kill about a half of the cells. Here I have a few questions.

1) Should I raise my G418 selection concentration? Because some people told me that the right selection G418 concentration should kill all the untransfected cells on day 6.

2) The G418 concentration, which affect my cell growth, is way much higher than that most of you guys were talking about in the forum. Why are my cells so resistant to G418?

3) If I use a selection G418 concentration more than 2mg/ml, does such a high concentration of G418 affect the transfected cell growth too (I will use pCDNA3 vector)?

Any comment will be greatly appreciated!

Are you calculating your G418 concentration from the active concentration listed on the bottle? HEK should be selected 800 ug/ml and maintained 400 ug/ml.

I agree with tap14.

I feel the G418 that you are using is losing activity or that you have calculated the concentration wrong.

800ug/ml is sufficent usually for selection in 293 cells.

Thanks, guys. The G418 was supplied in solution at 50mg/ml active concentration by the manufacturer. I recalcuted the G418 concentrations I used for the death curve, and I am sure my calculations were right. I think there must be something wrong with my G418 (actually it was new when I started the death curve experiments), as you guys mensioned, losing activity. By the way, is G418 sensitive to visible light too?

its sensitive to UV light (info from kosmodrom)

I've found that G418 solutions that are sterile filtered have much lower killing activities per ug/ml. Both that I have bought from companies or have personally sterile filtered. Perhaps that could be your problem? Or perhaps you just have a bad lot.

Hi Mountainman,

Thank you for your note. You have a point, I think. In my posted experiments, I used the G418 solution bought from Invitrogen. Now I am redoing the experments with G418, which I made from powder and sterile filtered. Now it is the 5th day of new G418 treatment, I don't see any sign of cell death at 2mg/ml G418. I think I will try the G418 without filtering. But how did you avoid contamination without sterile filter? Can it be autoclaved? Thanks.