Luciferase and cell responsiveness
Posted 21 January 2011 - 08:50 AM
I have transfected my cells with a luciferase reporter vector in two different cell types. On one cell type I am getting an RLU (over control) of 30; and in the other it is 300.
Now here is my problem, in both cell types, when I stimulate with LPS, LTA or heat killed bacteria, I get a reduction in levels of luciferase (The gene promoter is IL8).
This doesnt make sense to me. It looks like my transfections are working well. Do you think that both cell types are unresponsive or is there something in the media that is maximally stimulating my cells and therefore they do not have any extra capacity to respond?
Any thoughts would be greatly appreciated.
Posted 21 January 2011 - 09:14 AM
How long are you waiting to take the reading after addition of the stimulants? It is hard to konw if your stated RLUs are high or not since I don't know the background, but you could have basal actiivty in the conditions you are using or the cells could be dying as a result of treatment. If you continue to have issues, please do not hesitate to contact Promega Technical Services.
Posted 24 January 2011 - 11:05 PM
The other thing you need to check is transfection related toxicity. Check under microscope see if cells look alright.
Posted 26 January 2011 - 01:13 AM