Hi there,

I would like to go about doing a kill curve on my untransfected cell line. However, I am a bit confused as to how to carry this out. So I have a number of questions:

1. What type of plates do you normally carry this out on? What type of cell concentration should these cells be at? I ask this because it says that G418 is less effective if there are a greater concentration of cells. What is the optimal concentration of cells that should be in each well?

2. Is there any type of preparation that needs to be done to the G418? I.E. I have noticed that you can buy powdered G418 - what do you mix this with? Water? PBS? Media?

3. I read a number of different forums and websites that discuss how to create a kill curve, but I feel as if I got mixed information. I know that I want to subject the cells to various concentrations of G418. Do you suggest that I jump by 100ug or go up by 50? The average amount of G418 that has worked on related cell lines is 500ug/ml. Are you going to see that great of a difference in cellular lifespan when subjecting the cells to 500 vs 400 or 600ug/ml?

4. After I have my cells in the wells and I make up the different concentrations of media+G418 and add it to the wells, how long do I wait before I change the media. 1 day, 2 days? Then do I change it every day?

5. Different websites and forums have said that you use the lowest concentration of G418 that killed all of your cells in about 6 days. They then go on to state that you should wait about 2 weeks to make sure all cells have died. This is confusing because aren't all of your cells in that well already dead?

6. Do you decrease the amount of G418 after 2 weeks of selection by half? Meaning, if it took 500ug/ml to kill all cells in 6 days, do I maintain stably transfected cells in 250ug/ml for the rest of their lifespan?

So I guess overall, I plate my cells, let them grow to X% confluency. Add media+varying amounts of G418. Refeed meda+G418 either every day or every other day. Look for 100% cell death in the lowest amount of G418 added after 6 days. Give it a few more days to make sure that no cells were missed.

Ex. Let's say that 400ug/ml some cells still lived. 500ug/ml, cells were all completely dead after ~6-7 days. 600ug/ml cells were completely wiped out at 3 days.

So with this result I would assume that it is safe to use 500ug/ml on my cell line after I complete the transfection. And so, after transfection, I will subject the cells to G418 concentrations at 500ug/ml. After 2 weeks I can then decrease the concentration of G418 in order to maintain the cell line. Do I have the right frame of mind?

Thanks for any help!

Actually, I want to ask the same question.