Optimizing the ISRE-SEAP assay - (Aug/10/2009 )
I'm currently trying to get the secreted alkaline phosphatase to work for me. I have a construct where SEAP is induced by type I and III interferons (through ISRE). I transfect my human cell line with this vector (1 ug of DNA/2,5ug lipofectamine for each well in a 12-well tray). However, even when I don't treat my cells with interferon I still see a lot of activity compared to the treated cells. We have tried to shorten transfection time so we transfect for 8 hours, wash the cells in PBS after transfection, add media to cells, exchange media after 5 hours and then treat with interferon for 24 h, and then do the assay.
Do you have experience with this assay or general knowledge of how to limit interferon activation of the cells?
It sounds like your plasmid is leaky - there isn't much you can do about it unfortunately.