Hello;
I doing a killing curve for G418 disulfate salt, (((( I wonder what's the difference on just G418 or g418 sulfate,, I really dont know which one to use for stable cell lines, but I bought the G418, disulfate salt from Fisher, any suggestions?? ))))

The point is that my cells are not dying even with 900ug/mL, and they've been sitting for 4 days, I've been replacing the media every 2 days and nothing, they are just fine. I don't know if its that I have to many cells, ( I plated them in a 12 well plate, and they are pretty much confluent, like 90%)

What am I doing wrong?? Any suggestions in how to make a good killing curve??
I am using SKBr3 cells, breast cancer cell line.
thanks


So, I read filtering the G418 solution lowers its activity, and I did filtered, so, any comments in how to do a sterile stock without filtering?? I bought mine as a powder.

thanks

I've been having similiar troubles as well with a bowel cancer cell line. I had cells that would seem to be going okay at 1500ug/ml. I am going to repeat these experiements though because I made my G418 stock too dilute, which meant I was adding something around 100ul to a 1ml culture plate well. I think the effect I was seeing was actually 'drowning' of my cells rather than the action of the G418. This itself is a bit of a worry because usually a massive concentration would kill the cells, regardless of whether the cells were 'drowning' in diluted media or not. ie the concentration I'm using of G418 may need to go higher again.

I don't know a great deal about G418, maybe someone else could clarify - does G418 kill non-dividing cells or does it stop the non-resistant cells from growing and leaves the resistant cells untouched? In that case you would need to grow for a while so that the resistant cells can out grow the non resistants which will eventually die off of old age I guess you could say. Not sure if thats what happens but perhaps it may explain the results a lot of people see?

Good luck with it,
Dave

Heheheh,, Finally someone said something about it. I have the problem as well regarding the stock solution, I hade it to diluted so I'm adding a lot, but I also have a water only control just to make sure im not drowning my cells. My stock is 9.5mg/mL which normally people use higher concentration as stock.

wait for a week to see the complete effect of G418. Also many times G418 loses activity and one has to make up fresh G418. I did try solutions only have lower activity, so I made it up from powder and this works.

search G418 in the forum and you will find many posts regarding this topic.