I met a problem in my experiments with TNF-a inhibitors. I am testing different compounds for ability to inhibit TNF production, I have checked RAW264.7 cells, human PBMC, human macrophages, mouse splenocytes. I add inhibitors 1 hour prior LPS treatment and check TNF-a after 4 hour of incubation. In all cases TNF-a was inhibited in dose-dependent manner and I could generate IC50 graphs easily. This time I am testing mouse adipose tissue culture (because this experiment is closer to physiological conditions) and my results are very strange. I cannot generate IC50 graphs because almost all concentrations of inhibitors induce the same level of TNF-a decrease. even the lowerest concentration 0.1nM induce decrease of TNF-a production although in experiments with single cell suspension the minimal dose which is able to inhibit it was about 0.16uM-0.032uM (that dose had no effect on TNF production). So I got the graphs like gorisontal line with no dose-dependent realtion.. and cannot define the dose with no effect..
may be anyone has an idea why results are so different in single cell suspension and tissue explants??
I can find only one explanation yet: the cell number in tissue is much lower than in single cell suspension so may be even a smallest dose of inhibitors in enough to induce inhibiton of TNF.. I tried to increase tissue culture concentration in 2 time but still got the same result - no differences between all 10-fold dilutuions of inhibitors (5 10-fold dilutions show the same level of inhibition).
I would appreciate any advice and help!