Figure 9.

The effect of rIFN-γ or vdIFN-γ in combination with a TLR2/1 ligand on macrophage activation was detected using growth inhibition assay in Lewis lung carcinoma (LLC) cells. (A) Schematic timeline of the experiment. Mitomycin C-treated bone marrow-derived macrophages (BMDMs) were plated at three different densities and incubated for 24 h before macrophage-stimulating factors were added. After an additional 24 h, 100 µL of cell culture media was obtained from the wells with the highest macrophage density to measure ${\text{NO}}_{2^{-}}$ levels. LLC cells were added to the activated BMDMs, and the co-cultures were incubated for an additional 20 h. [³H]-thymidine was then added to the co-cultures, and after 24 h, the LLC cells were harvested to analyze cell growth by measuring the amount of incorporated [³H]-thymidine as counts per minute (cpm, depicted on the y-axis). (B) LLC cell growth after co-cultivation with BMDMs that were activated with Pam3 (100 ng/mL) in combination with different concentrations of rIFN-γ ranging from 100 to 0.0015 ng/mL in fourfold dilutions. Controls, from left to right: BMDMs cultivated without LLC cells, LLC cells co-cultivated with unstimulated macrophages, and LLC cells co-cultivated with macrophages that were stimulated with either rIFN-γ alone or Pam3 alone. (C) LLC cell growth after co-cultivation with BMDMs that were activated with Pam3 (100 ng/mL) in combination with vdIFN-γ ranging from 100 to 0.0015 ng/mL in 16-fold dilutions. Controls, from left to right: BMDMs cultivated without LLC cells, LLC cells co-cultivated with unstimulated macrophages, LLC cells co-cultivated with macrophages that were treated with either rIFN-γ, vdIFN-γ, or Pam3 at a concentration of 100 ng/mL, and LLC cells co-cultivated with BMDMs that were treated with 100 ng/mL of both rIFN-γ and Pam3. Experiments were performed in triplicates and were repeated two times with similar results. The bars represent the mean values of triplicates ± SEM from one representative experiment.