Measurement of macrophage cytokine production
Monolayer cultures of FLDMs and apoptotic thymocytes were prepared as described above. FLDMs were incubated with medium, LPS (10 ng/ml), apoptotic cells (ratio 1:10) or both for the determination of IL-10, TGF-β1 or TNF-α levels after co-culture for 22 h. For TNF-α quantification at various time points, FLDMs were cultured with a high concentration of LPS (100 ng/ml). Culture supernatants were harvested and TNF-α (Mouse TNF-α OptEIA set; BD Biosciences, Heidelberg, Germany) and TGF-β1 (Quantikine, TGF-β1 immunoassay; R&D Systems) were measured by ELISA as described by the supplier. IL-10 in culture supernatants was determined by a cytometric bead assay (Mouse inflammation CBA; BD Biosciences) as indicated in the manual. Data are presented as mean ± SEM from at least three independent experiments, each carried out in triplicate. Analysis of the results used the Wilcoxon-signed rank test; p values below 0.05 were considered significant.