When you say "antibiotic selection was successful" I assume that your cells grow normally in flasks?. If this is the case you need to avoid rapid change in the medium and try to first grow the cells in the same medium of your AB selection then slowly introduce any change that you want to make in the medium.

However, if your cells do not grow at first resuspension and plating, it means that your AB selection was either not successful or you didn't give it enough time to select only positive cells. Therefore, you have collected both negative and positive cells if there was any. In that case you might've had zero or very limited number of positive cells on your new plates and they were not enough to re-grow.

I would suggest that you give a longer time frame (2-3 Weeks) to the AB selection and change the medium every 2-3 days following 2x PBS wash so as to only collect the positive cells. On the other hand, if non of your cells survive it means that your transfection is not working and you need to either optimise your transfection protocol or change your transfection reagent.

Good luck.

Hi Denise, what types of wells or plates are you trying to grow your single cells in? Typically, as you probably know, limiting dilution and plating 1 to 0.5 cells per well in a 96-well is performed for such aims. If you aren't using 96-well plates, it might improve your results. Single cells don't like to be alone, the smaller volume of a 96 well plate helps them condition the media and do what they must to survive (in my opinion). Also, if selection was complete, you may try removing the selection agent altogether until colonies form, then add it back. You may lose some colonies, but it will provide the least amount of stress to those single cells you're attempting to expand. One last thing, you might try using conditioned medium from a larger culture to grow the single cells in once you plate them. It might help them be less stressed. Good luck.

Hi Erik, Thank you for your kind reply. Yes, I am using 96-well plates, one cell/well. I will try removing the antibiotics when starting the single cell culture. Thanks!!

Dear Soran, Thank you for your reply. I did the transfection in flasks and confirmed by SDS PAGE. Then, I tried 2 strategies (it took about 3 weeks). 1. Antibiotic selection on flasks and limited dilution in 96 well plates (cells did not attach). 2. Antibiotic selection on 10 cm dishes, pick up colonies and transfer to 96 well plates (colonies did not attach). I will tried removing antibiotics before single cell/colony culture. Thanks!

Denise, are you growing the ExpiCHO cells as adherent cells in T75 flasks? Do they seem happy if so? Just curious. From my impression they were designed to grow as non-adherent cells but I haven't read the manual. Have they been attachign well for you? I ask because you mention they are not attaching and that this is your indicator of an non-viability issue.

Hi Erik, for routine culture and transfection I use suspension culture. I just tried plating on 10 cm dishes for antibiotic selection and it actually worked, after a couple of weeks some colonies grew. However those colonies did not attach after passing them to 96 well-plates.

condition medium