Intriguing indeed. I strongly suggest to have a control- a cell line for which puromycin concentration that kills all cells in 24 h is known. This will tell you whether the puromycin that you are using is of expected potency. If not, there are other possible, but unlikely, explanations.

How often did you monitor the cells? Was it just 1 measurement after 24h? And are these pretty rapidly growing cells?

I wonder if you've selected out some resistant clones in the population that have then rapidly grown to confluency. You might expect to also see this occur in the 2.5ug/mL and 1.25ug/mL wells if you continue to monitor them but it might just occur more slowly because the drug concentrations are lower. Maybe you "overshot" the stage where the 10ug/mL and 5ug/mL populations appeared to be completely dead and are now only seeing the end result of selecting for resistant clones. If you checked on your wells every few hours, you might see an initial drop-off and then a recovery of the culture.

Hi Oliver,

it's definitely weird that you see cells surviving 10ug/ml while dying between 1-2.5 ug/ml.

You need to be more specific on how you did what you did, so I am not sure my thoughts will help you:

- are you seeding the cells in each well at the same numbers or are you seeding fewer cells in the low puromycin concentrations? The higher the confluence, the less effective the drugs are. So if you are making a solution of cells in 10ug/ml and then diluting it with medium, you not only dilute the puro but also your cells.

-What type of cells are you seeding? And is this a parental cell line, e.g. is it possible that your cells are a clone of a previous stable cell line generation with puromycin resistance?

To perform a killing curve, I normally seed cells at 30-40% confluence and evaluate which drug dose will kill all the cells in 3 days (not just one).

Hi Oliver,

Its really weird...!!!

I have use puromycin for generating stable clones in 5 different cell lines including the most sensitive NSCs to most tough cells HepG2. The range of puromycin for me was 0.1ug to 1.25ug/mL of media.

Even more than 2ug is rare and for very few cell lines. First you should check the puromycin stock, its efficiency vary from lot to lot. Puromycin is a very stringent selection marker it can kill most of the cells within 24 hours at more than 2ug/mL concentration. 30ug....!!! Something weird is happening.

When you are doing Kill curve I will suggest not to use 96 well plate instead use 24 well/12 well plate to avoid dilution based human error. Take the early passage parental cell line for the kill curve experiment. Add the Puro-media at 24hrs of cell seeding when its ~40% confluent. Change the media in every alternative day to avoid excess load of dead cells and to maintain the efficiency of puromycin at 37C. Selection concentration is the one kill ~50% cells in 2 days (2 doubling time and for puro strictly).

This may help you.

Good Luck

The similar thing is happening to me! Do you have any updates after that? I am suspecting that I have not mixed my puromycin solution well and plan to redo all.