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Setting up two drug kill curve for selection of stable cells

Kill Curve Stable Selection G418 Puromycin

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#1 Epigeneticist

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Posted 27 March 2014 - 05:37 AM

Does anyone have a recommendation for determining what concentration to use for selecting stable clones with two drugs such as G418 and Puromycin - dual selection instead of a sequential. I have already determined the concentration for each drug alone in my cell line. Do I need to perform a kill curve with the drugs combined together? These are common drugs so I am sure someone has experience with this. Didn't know if the combination would be more lethal and require a lower combined concentration. I thought about doing a kill curve. However, the confusing part is if I need to vary one drug concentration and not the other. Plus its hard to determine if both drugs are causing the effect or if only one of them is which is an issue selecting clones with both resistance markers. 

 

Thanks!



#2 bob1

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Posted 27 March 2014 - 11:36 AM

So long as there are no synergistic or competitive effects (i.e. work through different mechanisms and don't help each other) then the curves done independently should be enough.  The way to test for synergy and competition is usually to get a disc impregnated with the drugs, place them onto a soft agar overlay on a confluent layer of cells, then look at the shape of the kill zones.  If they are not round zones, then there are either synergistic (zones bulge towards each other) or competitive (zones flattened on sides nearest the other drug).







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