Stable cell lines are a crucial laboratory tool that over-expresses a gene of interest in order to study gene functions, screen experimental drugs or produce therapeutic proteins (i.e. recombinant antibodies). The cell lines will divide and continue to express the inserted transgene. Briefly, exogenous plasmid DNA is transfected into a host cell line, which is followed by antibiotic pressure to select only those cells that integrated the exogenous DNA.
Prior to beginning the study, a kill curve for the antibiotic and cell line should be established. This is performed as follows:
- Plate the cells at typical passaging ratio (e.g. 1:5, 1:10) into the plate or flask to be used in stable cell line creation. To the culture medium add concentrations of the antibiotic in a dilution series. Each antibiotic has its own working range concentration and needs to be established. As an example, Puromycin is commonly used at concentrations in the range of 1-7 µg/mL for adherent cells. This is a general guideline and is dependent on each cell line.
- Incubate for 7 days, replacing the antibiotic-containing medium every 4 days.
- Count the number of viable cells in each well.
- Plot the number of viable cells versus the antibiotic concentration. This curve establishes a kill curve to determine the antibiotic concentration required to kill cells that did not integrate the plasmid DNA. The optimal dose is the lowest antibiotic concentration where all cells are dead after 7 days. Higher doses will kill all cells after 2-3 days, and low doses will have minimal toxicity after the 7 days. Use the established optimal concentration for stable cell line selection.
We recommend using 200 – 800 ug/ml of Neomycin (also called Geneticin or G418) for mammalian cells selection. While HeLa cell is efficient at 200 ug/ml of G418, other cell lines require higher concentration of G418 (for example, optimal G418 concentration for A549 cell line selection is 800 ug/ml). Typical G418 concentrations required for indicated cell line selection:
Please note that mammalian cells selection requires use of puromycin, geneticin (G418), zeocin, hygromycin, or blasticidin. Ampicillin (Amp) or kanamycin (Kan) are not effective for mammalian cells selection in tissue culture experiments (Amp and Kan can only be used for bacterial selection).