IEC-6, IL-6 and LPS - Cannot get my IEC-6 cells to secrete Il-6 (Oct/28/2008 )

Help! i am trying to get my IEC-6 cells to secrete Il-6 after stimulation with LPS, and i am quantifying by RAT Il-6 ELISA kit (BD biosciences).

Basically, i add LPS (range of 0.5-10 ug/mL) in growth media (with serum, antibx, insulin) to my IEC-6 cells in 12 well plates for 3 days. At the end of three days i collect the supernatant and spin down and do the ELISA.

my standards from the kit work beautifully, although i do not have any other positive control.

does anyone have any suggestions? i think i'm following basically what i've read from other people's papers, and i cann't figure out what i'm doing wrong. do i need to worry about mycoplasma contamination, or looking for other obscure reasons?

the only other thing that i just realized i'm doing wrong is that according to the kit instructions, i should add the detection antibody first for an hour, then the y HRP for half an hour - i've been combining them both, and adding them at the same time to my wells (this is what they instruct in their human il-8 kits). since my standards are working, i don't think this is really the problem, though. however, i've done this experiment twice now, and i'm a little reluctant to do another expt without some advice, as the kits and the ligands are very expensive!

thanks for any help!

hi Luna rota,

You can try to collect supernatant at different times, in three days of incubation with LPS (with your specific cells, with your specific lot of LPS) you can have a peak IL6 in few hours and, when you are measuring IL6 after 3 days could be that IL6 is degraded. Cytokines are labile molecules.

I hope that you serve