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1. Are cell density (% confluence) and passage number important considerations for transfection?

Yes, cell density is an important parameter in influencing transfection efficiency. If the seeding density is too low, some cytotoxicity may be observed. If the cell density is high, lower than expected transfection efficiency may be observed. Both issues may be easily resolved by either decreasing or increasing the quantity of complexes added to the culture. LipoGeneTM provide excellent transfection efficiencies at confluence between 70 and 90%. Some toxicity may be observed at lower confluence but may be alleviated by decreasing quantity of complexes or removing the complexes after 4-6 hours incubation and refreshing the media.

Passage number may affect transfection experiments. Excessive numbers of passages may decrease transfection performance. We do not recommend splitting cells for more than 20-30 passages. If transfection performance declines and cells have been in culture for a long time or excessively/improperly passaged, we recommend that you restart your cultures with a new vial of cells from liquid nitrogen.


2. Can antibiotics be used in media during transfection?

Yes, antibiotics (penicillin-streptomycin) can be used in the medium during transfection. We have compared transfecting cells in medium with and without antibiotics in multiple cell lines, assessed both the transfection efficiency and toxicity, and found no difference. For certain cell types that are sensitive to transfection or have toxicity issues, omitting antibiotics may improve results, however. For stable transfections, wait at least 48 hours after transfection before adding selective antibiotics.


3. Why would the expression level of my gene in transiently transfected cells be greater than those that are stably transfected?

Expression in transiently transfected clones is typically higher because transiently transfected cells can have up to hundreds of copies of the plasmid containing the gene of interest. Stably transfected clones usually harbor 1-2 copies integrated into the genome, and hence have lower levels of expression. Sometimes, the lower expression level in stably transfected cells is due to adverse effects of the recombinant protein on the cell when expressed constitutively.


4. Can I use the same amount of any transfection reagent for different cell lines?

The transfection efficiency is highly dependent on the amount of reagent used per well and may be different between reagents. Please consult the product information that is provided with the transfection reagent for optimal use.


5. Can I use LipoGeneTM for transfection of suspension cells, other hard-to-transfect cells, and primary cells?

Yes,you can use LipoGeneTM for transfection of suspension cells, hard-to-transfect cells, and primary cells