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Determining Antibiotic Killing Curve for Stable Cell Line Generation


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Determining Antibiotic Killing Curve for Stable Cell Line Generation

When generating a stable cell line in mammalian cells, if the expression system carries a drug resistance gene, it is an efficient way that stably-transfected/transduced cells are selected by the addition of antibiotic drugs to the culture medium. Since mammalian cells sensitivity to antibiotics varies from one cell type to another, it is essential to determine the minimum concentration of antibiotic required to kill non-transfected or non-transduced cells by separate kill curve experiments.

Protocol

1. Plate cells in 0.5 mL complete medium per well in 24-well plate. Ideally, cells should have reached high confluence prior to adding the antibiotic. Typical cell density ranges are as follows:

  • Adherent cells: 0.8 - 3.0 x 105 cells/mL
  • Suspension cells: 2.5 - 5.0 x 105 cells/mL

2. The next day, replace growth medium with selection medium supplemented with a range of antibiotic concentrations.

Notes:

  • A no-antibiotic control is included.
  • Maintain each concentration in triplicates.
  • The dose response may vary with the cell line being tested and the antibiotics.
Selection AntibioticConcentration Range
Puromycin0.25-10 μg/mL
G4180.1-2.0 mg/mL
Hygromycin B0.1-0.8 mg/mL
Blasticidin1-20 μg/mL

Table 1. Recommended concentration of antibiotics for mammalian cells

3. Replace medium containing antibiotic drug every 2-3 days. Check cell death every day by light microscopy.

4. The minimum antibiotic concentration to use is the lowest concentration that kill 100% of untreated control cells in required time (~7 days).

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