I have recently started working with HEK293 cells. I am trying to measure NFkB activation in these cells by measuring luciferase activity. I co-transfect HEK293 cells with TLR and reprtor plasmid (it had expressing renilla luciferase enzyme under the promotor of NFkB) and then stimulate cells with TLR specific ligand. Afterwards I measure lucuferase activity using Promega kit.
So, I have two questions in relation to my work.
1. I normally get quite high background relative light units for my control. Is that normal (If someone is doing same kind of assay)?
2. What is the benifit of co-transfecting HEK cells with reporter plasmid (in my case pRenilla) and measure luciferase activity over measuring IL-8 by itself (in HEK cells)? If this assay is just to measure NFkB activation then why not IL-8, as HEK293 cell will produce IL-8 only if NFkB is activated otherwise no IL-8 and no NFkB. By doing so one does not has to co-transfect HEK cells with reporter palsmid and transfection with only receptor encoding plasmid will be enough and measure IL-8 production.
3. And what is the real point of measuring luciferase activity after transfecting cells with plasmid which contains renilla expressing gene under the promoter of NFkB? At the end of the day NFkB will switch on the cytokines production so why not end product.
I might be missing some important point that’s why I have asked these question.
So, if someone can help me.
Kind regards,
Sandeep
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