Antibiotic Selection Optimization Using a Cytotoxicity Profile

Protocol provided by the MISSION® team


The appropriate concentration of antibiotic for selecting stable cell lines is different for each cell type. If the concentration for the desired cell type is unknown, a titration experiment must be performed to determine the lowest concentration of antibiotic needed to efficiently select transduced cells. In this protocol, we highlight the use of puromycin, the antibiotic utilized with the standard pLKO.1 vector. Typically, 1-10 µg/mL are sufficient to kill most untransduced mammalian cell types. This general protocol can also be utilized for determining the optimal concentration of G418 required for the custom shRNA vectors. Higher concentrations than absolutely required of antibiotic could lead to off target effects and fewer cells for downstream analysis.


  • Cells - in log growth and at 50% confluence on the day of transfection
  • Cell Culture Media
  • Tissue culture incubator—37 °C, 5% CO2, 100% relative humidity
  • Antibiotics puromycin (P9620) or G418 (A1720)



If cells started to round but did not detach from the plate surface after addition of puromycin:

  • Rounding could be a sign of cells dying without detaching from the surface yet. These cells should detach with more time.
  • If cells require higher concentration of puromycin then the recommended 1-10 μg/mL, check the expiration date on the puromycin and avoid multiple freeze-thaw cycles (<5).