We use Hek-Blue-TLR4 (TM) cells from Invivogen.

They are substantially Hek-293 cells transfected with TLR4, MD-2 and CD14 genes and with a reporter gene for a secreted alkaline phosphatase that turns on when NFkB signal pathway is activated by TLR4/CD14 agonists. A colorimetric reaction with the Hek-Blue phopshatase substrate (Invivogen) or the much cheaper p-nitrophenylphosphate gives a quantification of the alkaline phosphatase produced that is proportional to NFkB activation (and since no other TLR is expressed in the cells also proportional to TLR4 activation). A proper negative control cell line (Hek-null) is to be used.

Why don't you take a look at some of our publications (Francesco Peri - Matteo Piazza)? Also, if you are interested in a collaboration (you send the sinthetic compounds and we test it in our cell lines) let us know.

Best Regards

Roberto Cighetti, University of Milano-Bicocca

Hi Roberto,

Thanks for your answer! Finally I came to the same result, working with Invitrogen HEK-Blue cells. Since I have no experience with this system, I am wondering, whether it is sensitive enough. From what you write, it seems to work.

Thank you very much for your polite offer for cooperation. At the moment I have no substance to be tested. I need TLR4 activation as a measure of inflammation. Perhaps there is an opportunity for cooperation in future.

Kind regards, Kurt

I have used the cell line for TLR4 activity, its a nice assay. But this cell line has some endogenous expression of TLR3. 5 and NOD1. I need an alternative approach to measure the TLR4 activity. Any suggestion ???