Applications: Stable Cell Line Generation

Determining the Optimal Selection Antibiotic Concentration

The first critical step for stable cell line generation is determining the optimal antibiotic concentration for selecting stable cell colonies; the optimal concentration is cell type dependent. A kill curve is a dose-response experiment where the cells are subjected to increasing amounts of antibiotic to determine the minimum antibiotic concentration needed to kill all the cells over the course of one week. Performing a kill curve is recommended with each new cell type or when a new selection antibiotic or different lot of selection antibiotic is used.

  1. Plate cells in 0.5 ml complete growth medium per well in a 24-well tissue culture plate one day prior to introducing antibiotic selection. Ideally cells should have reached high confluence (~60-80%) prior to adding the selection antibiotic. Typical cell density ranges are as follows:
    • Adherent cells: 0.8–3.0 × 105 cells/ml
    • Suspension cells: 2.5–5.0 × 105 cells/ml
  2. Add increasing amounts of the appropriate antibiotic such as G418 to duplicate wells of cells plated in complete media. Include a no-antibiotic control. For example, add 0, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 µg/ml selection antibiotic to duplicate wells of cells plated in complete growth media. Please refer to the antibiotic specific range of concentrations below.
    Selection Antibiotic Working concentration range
    G418 0.1 -2.0 mg/ml
    Hygromycin B 100 - 500 µg/ml
    Puromycin 0.25 - 10 µg ml
  3. Replace media containing selection antibiotic every 2-3 days for up to a week. Examine the culture every day for signs of visual toxicity. Determine the following antibiotic doses:
    • Low dose - the antibiotic concentration at which minimal visual toxicity is apparent even after 7 days of antibiotic selection
    • Optimal dose - the lowest antibiotic concentration at which all cells are dead after one week of antibiotic selection
    • High dose - the antibiotic concentration at which visual toxicity is evident within the first 2-3 days of antibiotic selection
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G418 Titration for Determining Working Selection Concentration

G418 Titration for Determining Working Selection Concentration. Increasing amounts of the antibiotic such as G418 (0-1000 µg/ml) is added to duplicate wells of cells plated in complete media in a 24 well plate. Visual toxicity is assessed for upto a week to determine low, optimal and high doses for antibiotic selection.

For stable cell line generation, use Mirus' high quality cell-culture grade selection antibiotics that are easy-to-use:


All Mirus' broad spectrum plasmid DNA transfection reagents can be used for stable cell line generation. Visit the product pages for more information on each product:

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