Help with IEC-6 ELISA - (Jun/28/2010 )

I am using IEC-6 cells cultured in 6 well plates overnight in their growth media (dmem, insulin, fcs, p/s) and am stimulating with lps (10ng-1000ng/ml) and have been attempting to quantify il-6 levels at different time points - 1 - 24hr.

I have been using the BD rat 1l-6 elisa kits but have gotten nil response....is there something i am doing wrong? my standards are working out with R factor .97

Please help!!

Whts the exact conc of LPS used? Whts tht no response.. is that showing equal value compared with the blank?
After collecting the culture soup u r doing the elisa immediately or storing the samples.. So which temp u r storing?
How you are preparing samples for the asssay.. ?
1ug/ml is more than enough to stimulate... B/w 8-18 hrs will give maximum levels of IL6...

I am using three diff concentrations of lps - 10ng/ml, 100 ng/ml, 1000 ng/ml
I have a stock solution of 1 mg/ml and make a working of 10 ug/ml (10 ul stock in 990 of pbs) and adding into wells.

I aspirate the supernatants and store in -80 immediately and did the elisa within the next few days. I thaw at room temp and use in elisa.

I also had a plate that had no lps stimulation and i aspirated at the same time points.

The supernatants that had lps stimulation had the same od value (and conc) as no stimulation. Which was essentially nil.

At the later timepoints the cells were starting to get crowded, I did not change media prior to lps stimulation to remove dead cells etc, which i will try next time.

The standards work with a r value of .97

Thanks for the help