Primary bone marrow-derived macrophages (BMDMs) were derived from bone marrows of 6−10 weeks old C57BL/6 WT and Zbtb16-/- mice were prepared by methods described previously21. Briefly, bone marrow cells were prepared from mice and cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum (FBS), 30% L929-conditioned medium and antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin). After 4 days of culture, nonadherent cells were removed and a fresh culture medium was added. On day 6 cells were seeded into 6 well plates at 1.5 × 106 cells/well for experimentation on day 7. Immortalised WT and Asc-/- BMDMs (gifted by Dr Eicke Latz, Institute of Innate Immunity, University of Bonn) were cultured in DMEM supplemented with 10% FCS and 10 μg/mL Ciprobay-500. Peritoneal macrophages were collected by lavage with 10 mL RPMI-1640 medium. The cells were subsequently plated in 96-well plates and cultured overnight for further study. HEK293T, HeLa and THP-1 cells were purchased from ATCC and were grown in DMEM (HEK293T and HeLa) or RPMI-1640 (THP-1) with 10% FBS with antibiotics. All cells were cultured in a 5% CO2 incubator at 37 °C.
