if treated and not treated cells give you the similar numbers there are few reasons:

-it could be that the cells already have mycoplasm so unstimulated cells show already high levels of cytokines. This is likely the case if you already have high levels of cytokines of unstimulated cells.

-are you starving the cells?

-the cells do not respond to the treatment (whatever the reason might be- no receptor or particular agonist, old chemicals...)

-If it is commercial ELISA, you can always phone the company for troubleshooting. First make sure that your values are in the linear range on the standard curve.

Hope this helps

Thanks for the information. I guess the reason is the cells. I have tried with different ELISA kits and i got the same results in several runs.

Dear Omid,

Some ideas :

- Which time points are you following, after activation ? IL6 might be very fast (in the first hours)

- some culture conditions seem to activate macrophages (such as certain plastic of the culture plates) - it comes from discussions long time ago, I don't remember who was telling me that, but it might play a role.

- can you check some activation markers to check that your macrophages are resting before stimulation (I'm not from the field so I don't know about them)

Best,

Dear Philippe 

I am determining immune responses during early time point, but i also have included 24 hour p .infection since i believed it might be representative of the second wave of cytokines.