3 replies to this topic

[KMeade]

Hello,

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.

[KevinK]

Hello,
   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.

Kind Regards,
Kevin

[genehunter]

Cell responses to LPS stimulation are only transient by nature. Be aware of the timing you harvest cells from the point of treatment starts. The response could go up then come down. Do a time course if I were you. Transcriptional factors like NFKb is activated 30 min after treatment starts and only remains for a couple of hours, then if fades away.  Then you need to give cell some time to make luc enzyme after transcription step. Do the math and plan for a time course.

The other thing you need to check is  transfection related toxicity. Check under microscope see if cells look alright.

[KMeade]

Thanks for feedback - I am wondering if endotoxin contamination of my vector is the issue. I hadnt used an endo-free kit, but am getting one in now. Although you would still expect to see a synergistic effect when extra LPS is added surely?