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Transient transfection trouble- massive cell death - (Jan/08/2009 )

I'm trying to transiently transfect a few constructs in order to evaluate whether N or C terminal tags would be best to use for a stable transfection. I have been using lipofectamine and I am using the Invitrogen plasmid pDEST53 as well as a control well with their control plasmid- GW-CAT-pDEST53. I have been doing a lipofectamine only well, a DNA only well, and then several wells with different combinations of plasmids to see if there is an increase in solubility of my recombinant proteins when cotransfected with each other. Anyways, I keep seeing massive cell death in all of my lipofectamine + DNA wells. There is no death in the DNA only wells and much less death in the lipofectamine wells. I am using a 1:2.5 ratio of DNA to lipofectamine.

Does anyone have any suggestions of why my cells are dying? Should I continue trying to optimize a lipofectamine transfection or try another transfection method?


Perhaps, it depends on how long you're leaving the Lipofectamine on your cells. Some cells are very particular and find that the Lipofectamine to be toxic if left on too long. The manufacturer (if memory serves) does mention something about removing the Lipofectamine from the cells and replacing with just regular media after some incubation time.
Just a thought...