BioLab

Hello,

I transduced my suspension cells with a vector containing antibiotic marker. 48 hours post transfection, I split the cells to remove them from RetroNectin plate.

My questions are:

1. What is the optimal cell number and volume to start with when performing the selection?
2. How frequent to add the antibiotic, and when adding it do I have to exchange the whole media?
3. Is it safe to split the cells during the selection procedure, or is it going to change the ratio of viable vs. dead cells?

My readout is PI staining, and the selection takes about 5-7 days.

Can you highlight what factors may contribute to rendering the cells resistance to the antibiotic (e.g. confluence culture, etc...)?

Thanks