G418 Kill Curve Protocol Background

G418 Kill Curve Protocol
Background
G418 disulfate, also known as Geneticin®, is routinely used to screen for resistant mammalian cells
which express the neo resistance gene. The neo gene encodes amino-glycoside 3’-phosphotransferase;
an enzyme which confers resistance to G418 disulfate. Before selection of resistant cells, an optimal
working concentration range needs to be determined.
The optimal working concentration of G418 disulfate for selection of resistant mammalian clones
depends on the cell lines used, the quality of G418 disulfate, media, growth conditions, cell density, cell
metabolic rate, cell cycle phase, and the plasmid carrying the neo resistance gene. A kill curve should
therefore be generated to determine the optimal working concentration for every experimental system
and for every lot of G418 disulfate.
Preparation and storage of G418 disulfate solution:
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Stock solution - Dissolve G418 disulfate in water at a concentration of 50 mg/ml.
Sterile filter the solution using 0.45μm filter.
Store solution at 2-8°C after use.
Method: Determine optimum working concentration for selection of mammalian cells
1. Plate cells in 0.5 ml complete growth medium (ex. DMEM + 10% FBS) per well in a 24-well tissue
culture plate one day prior to introducing antibiotics selection. Cells should be around 60-80%
confluent prior to adding G418 disulfate. Typical cell density for adherent cells is 0.8–3.0 ×
105cells/ml, and for suspension cells 2.5–5.0 × 105 cells/ml.
2. Add increasing amounts of G418 disulfate to duplicate wells of cells plated in complete media.
Make sure to include a duplicate control well with no G418 disulfate. General working
concentration for G418 disulfate is 0.2 mg/ml – 1.0 mg/ml.
3. Replace media containing G418 disulfate every 2-3 days for 7-14 days. Examine the culture
every day for viability of the cells.
4. Use the lowest concentration that gives 100% cell death after 7 days under G418 disulfate
selective pressure.