I currently work with some adherent cell lines, such as 293, which are grown in DMEM media. I have noticed that the mortality seems to be high after Nucleofection®. Do you have any recommendations?


For some adherent cell lines we strongly recommend using RPMI instead of DMEM medium. As described in culture conditions of the cell line optimization kit protocol as well as the NIH3T3 (DSMZ) protocol, using RPMI instead of DMEM as "transfer and recover" medium can be crucial for the survival of the cells whether you work with adherent or suspension cells. For some cell types, we see a very high mortality if we use DMEM immediately after Nucleofection®. Immediately after Nucleofecting your cells, we recommend that you add 500 µL's of pre-warmed RPMI+ 10% FCS to the cuvette and then remove the entire contents of the cuvette and place it into an Eppendorf tube at 37 C° for 5 -10 minutes to allow the cells to recover. After that time you can plate them in the appropriate media. There is no need to get rid of the RPMI, just add the cell suspension to the DMEM containing well. It is also possible to change the media after 5-6 hours.This will lower the mortality especially with 293 cells.