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Inducing apoptosis by puromycin selection: toxic to the selected cells?


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#1 seanspotatobusiness

seanspotatobusiness

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Posted 24 March 2014 - 04:57 AM

I have a question regarding selection of cells by treatment with puromycin. If the majority of cells apoptose, won't the medium become quickly toxic as the cell fragments degrade and spill? I figure I could overcome this by repeatedly replacing the medium but how often? I imagine this even more of nuisance if your cells are in suspension and need to be centrifuged every time.



#2 bob1

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Posted 24 March 2014 - 11:36 AM

Quickly is subjective - changing medium every 24-48 hours should be OK, and with puro the majority of susceptible cells will be dead within a few days.  The burden of dying cells will depend on your transfection efficiency, so somewhere about 70-50% of the vessel will die (i.e. 30-50% transfection efficiency), for most vessels this isn't a huge number of cells given that they should start at around 70% confluent.






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