I have been testing a NFkB luciferase reporter. I have always done it in HEK293 cells and the positive and negative controls are working fine. But as I changed to epithelial carcinoma cells, the luciferase is not being induced as it should be. As control for a working system, I measure as well the IL-8 cytokine production in the supernatant of the epithelial cells and I see that it is induced as my controls have always done, but the luciferase is almost none (Epitheal cells: RFU 600; HEK293 RFU 22000). A similar effect I saw with AP-1 luc (another transcription factor for IL-8 production) in the epithelial cells.
Could it be that HEK293 keep the transfected DNA somewhere else as the epithelial cell line? Has anyone had the same experience with other cells and luciferase reporters?
Hi Luisa,
you could have a problem with your transfection efficiency. Hek293 cells are generally much easier to transfect than most other cell lines.
I would suggest that you you transfect your epithelial carcinoma cells and HEK293 cells with a positive control reporter vector such as pGL3-CMV-luc (or a comparable vector of the next generation) and see if you get comparable signals. If not you must optimize you transfection protocol for your epithelial carcinoma cells.
Good luck,
Kai
Hi Kai,
thanks for your suggestion. At the moment I am using a pEGFP-N1 plasmid to see the transfection efficiency. With my other cells I get about 90% efficiency. With HEK293 about the same, so I don't think there could be the problem. I have the feeling that the plasmids are not equally accesible. While with the epithelial cell I can see the first EGFP in less than 6 hours, and in almost all of them, I get with the HEK293 the maximum of the fluorescence after 24-36 hours. What do you think? The idea of a CMV -luc will be still intersting to see if the cells are ptting the luciferase somewhere else. Thanks.
Until next time, Luisa
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