Iscove’s and RPMI medium were purchased from Biological Industries (Kibbutz Beit-Haemek, Israel); zymosan from Sigma-Aldrich (St. Louis, MO, USA). ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA) and used according to the manufacturer’s instructions. Apoptosis of murine thymocytes was induced by culture for 1.5 h at 37°C/5% CO2 in an RPMI medium of 600 irradiated thymocytes. Optimal conditions for thymocyte apoptosis without necrosis were selected, i.e.>60% cells bounded by Annexin V, but >95% excluded by PI and trypan blue, www.selleckchem.com/products/gdc-0068.html as described
earlier 12, 15. Cell cycle analysis following staining with PI was a second method to verify apoptosis 12, 15. Human macrophages were isolated from peripheral blood monocytes of normal donors, www.selleckchem.com/products/AZD0530.html as described earlier 12, 15. Briefly, monocytes were cultured on Chamber-Tek glass slides (Nunc, Naperville, IL, USA) in Iscove’s medium (Beit-Haemek Industries, Kibbutz Beit-Haemek, Israel), in the presence of 10% serum AB that was selected
after testing five to ten lots from different companies. The selection criterion was gradual morphological differentiation of monocytes to macrophages, which necessitated media replacement on days 3–4. At days 6–7, macrophages were fully differentiated and ready for interaction. The gold standard for such development was autologous blood sample of a healthy donor. Serum AB lots were excluded if, during the selection process, we noted that they caused accelerated differentiation and increased rates of apoptosis and metabolism, as judged by the color of the media.
We used the term nonactivated macrophages for macrophages that were generated using autologous serum or selected AB serum, and preactivated macrophages for those with accelerated differentiation using specific AB serum lots. For experiments with fibronectin, cells were seeded into wells coated with fibronectin (40μg/mL; Invitrogen, Carlsbad, CA, USA). Immature monocyte-derived DC were generated from the CD14+ selected fraction of PBMC, which were isolated using Ficoll Fenbendazole as described previously 8. Briefly, anti-CD14 magnetic beads were used to isolate monocytes from PBMC according to the manufacturer’s instructions (Miltenyi Biotech, Auburn, CA, USA). Monocytes were placed in wells at a concentration of 1.25×106 cells/1.5 mL culture media, in the presence of 1% autologous plasma, GMCSF (1000 U/mL), and IL-4 (500 U/mL). Every 2 days, 0.15 mL was removed, and 0.3 mL media containing plasma and cytokines was added. By day 6, >90% of the cells were CD14- and CD83-negative, with low expression of HLA-DR and CD86. Interaction between human macrophages and apoptotic cells was performed as described earlier 12.