Step	Problem	Possible solution(s)
Transfection	No reporter proteins evident or cells did not survive drug selection	Human error is the most common problem. Specifically, pipetting of the plasmid DNA. Use fresh media and a new batch of PEI. Cells density was too high.
Transfection	No survival after transfection or selection	Drug dose was too high. Human error is the most common problem. Specifically, pipetting of the plasmid DNA.
Transfection	Populations not expanding after > 7 days drug selection.	Drug dose was too high. Increase percentage of FBS in the media to 15-20%.
Protein expression	Selected cell populations not expressing desired protein.	If neither the reporter or POI are expressed, then check the expression vector construct to ensure the ORF does not have an early stop codon. Also confirm the expression vector sequencing was accurate and perfect. Some proteins will fail for unknown reasons. For well-characterized proteins such as IFN-γ, we expect this system to work > 95% of the time. For POIs that have not been produced recombinantly in mammalian cells, the failure rate is likely to be 10-20%.
Protein purification.	Low protein expressed/collected/purified.	Cells were not rapidly dividing in culture. Protein was degraded quickly in the supernatant.